Advances in Food Biochemistry

ADVANCES IN FOOD BIOCHEMISTRY ADVANCES IN FOOD BIOCHEMISTRY Edited by Fatih Yildiz CRC Press Taylor & Francis Grou...

Author: Fatih Yildiz

337 downloads 3319 Views 4MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form

DOWNLOAD PDF

ADVANCES IN FOOD BIOCHEMISTRY

ADVANCES IN FOOD BIOCHEMISTRY Edited by

Fatih Yildiz

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2010 by Taylor and Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number: 978-0-8493-7499-9 (Hardback) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http:// www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

Contents Foreword ..........................................................................................................................................vii Preface...............................................................................................................................................ix Editor ................................................................................................................................................xi Contributors ................................................................................................................................... xiii Chapter 1

Water and Its Relation to Food .....................................................................................1 Barçin Karakas¸ and Muharrem Certel

Chapter 2

Glycobiology of Foods: Food Carbohydrates—Occurrence, Production, Food Uses, and Healthful Properties ......................................................................... 23 Frank A. Manthey and Yingying Xu

Chapter 3

Amino Acids, Oligopeptides, Polypeptides, and Proteins ......................................... 51 Fatih Yildiz

Chapter 4

Enzymes Applied in Food Technology .................................................................... 101 Dimitris G. Arapoglou, Athanasios E. Labropoulos, and Theodoros H. Varzakas

Chapter 5

Lipids, Fats, and Oils................................................................................................ 131 Ioannis S. Arvanitoyannis, Theodoros H. Varzakas, Sotirios Kiokias, and Athanasios E. Labropoulos

Chapter 6

Nucleic Acid Biochemistry: Food Applications ....................................................... 203 Is¸ıl A. Kurnaz and Çag˘ atay Ceylan

Chapter 7

Hormones: Regulation of Human Metabolism ........................................................ 219 Ayhan Karakoç

Chapter 8

Physiologically Bioactive Compounds of Functional Foods, Herbs, and Dietary Supplements ................................................................................................ 239 Giovanni Dinelli, Ilaria Marotti, Sara Bosi, Diana Di Gioia, Bruno Biavati, and Pietro Catizone

Chapter 9

Flavor Compounds in Foods .................................................................................... 291 Dilek Boyacioglu, Dilara Nilufer, and Esra Capanoglu

v

vi

Contents

Chapter 10 Food Acids: Organic Acids, Volatile Organic Acids, and Phenolic Acids .............. 313 Y. Sedat Velio˘glu Chapter 11 Biological Oxidations: Enzymatic and Nonenzymatic Browning Reactions and Control Mechanisms ................................................................................................ 341 Fahrettin Gö˘güs¸, Sibel Fadilo˘glu, and Çi˘gdem Soysal Chapter 12 Lipid Oxidation and Control of Oxidation ............................................................... 383 Sotirios Kiokias, Theodoros H. Varzakas, Ioannis S. Arvanitoyannis, and Athanasios E. Labropoulos Chapter 13 Food Additives and Contaminants ...........................................................................409 Theodoros H. Varzakas, Ioannis S. Arvanitoyannis, and Athanasios E. Labropoulos Chapter 14 Nutrigenomics and Nutrigenetics ............................................................................. 457 Abdullah Ekmekci and Meltem Yalinay Cirak Chapter 15 Pharmacogenomics and Toxicogenomics in Food Chemicals ................................. 477 Bensu Karahalil Index .............................................................................................................................................. 497

Foreword Knowledge of food biochemistry is critical to the development and growth of major aspects of food science, including production, processing, preservation, distribution, safety, and engineering and technology. Each of these areas is directly related to the current effort of commercializing food products and adding new value to them. Putting this science into practice also requires an understanding of broader issues, such as national and global regulations, concerning the identity, manufacture, and transport of foods. Appreciating food biochemistry also offers insights into consumer perspectives and preferences regarding issues such as genetically modified foods, nanomaterials in foods, functional foods and nutraceuticals, and food safety. The successful application of this knowledge is ultimately essential to promoting health and wellness through food and nutrition. Advances in Food Biochemistry provides updated information on fundamental topics such as food acids, additives, flavors, contaminants, and safety. However, it is unique in that it covers topics on the emerging science of food glycobiology, plant and animal hormones, nutrigenetics, and toxicogenomics. While traditional food biochemistry texts deal with the major macronutrients and micronutrients, this book addresses the modern application of phytochemicals from plant foods and herbal medicines to functional foods and dietary supplements. Few books in this field have recognized the influence of nutrigenomics on food science, and the implication of the impact of foods and nutrients on the genome, transciptome, proteome, and metabolome. Applying this knowledge about the interactions between our genes, nutrition, and lifestyle presents an opportunity to develop new food products directed to optimize our health based on an individual’s unique needs. Similarly, toxicogenomics is covered here to describe not only its applications in deciphering mechanisms of toxicity, but also in aiding risk assessments. This emerging aspect of food biochemistry, including the use of microarray data, is now beginning to be used by government regulatory agencies to better predict harm and guarantee the safety of foods as well as drugs. Professor Fatih Yildiz has edited this book to provide a practical overview of foods from a biochemical perspective. He received his undergraduate education in Turkey and did his graduate training in the United States, where he served on the faculty at the University of Maryland, College Park before joining the Middle East Technical University, Ankara, Turkey. He has published an extensive body of research and worked on food and nutrition science projects with the FAO, UNIDO, UNICEF, and NATO. Most recently, Dr. Yildiz received the Ambassador for Turkey Award from the European Federation of Food Science and Technology. He has worked to ensure that each chapter emphasizes the nutritional and health aspects of food and its components as well as associated issues such as technology and toxicology. Dr. Yildiz and the other chapter authors have brought together their renowned expertise and decades of experience to this book, designing it for advanced college students who can use it to learn about this field without the need of additional references. Food science and policy professionals will also find this book a useful resource in their field of work. If we are to feed the world, books like Advances in Food Biochemistry will play an important role in leading the way. Jeffrey B. Blumberg, PhD, FACN, CNS Tufts University Boston, Massachusetts

vii

Preface This book is an outgrowth of my research and teaching to graduate and undergraduate students at the University of Maryland, College Park; the University of Minnesota, Minneapolis; the Institut National Recherches Agronomiques, Avignon, France; the Mediterranean Agronomic Institute of Chania, Crete, Greece; and the Middle East Technical University, Ankara, Turkey. The pace of discovery in biochemistry and its application to food biochemistry has been very rapid during the past decade. This increase in knowledge has enriched our understanding of the molecular basis of living systems as well as biomaterials and has opened many new areas of applications in food production, processing, storage, distribution, consumption, health, and toxicology of food and food components. Understanding the biochemistry of food and feed is basic to all the other research and development in the fields of food science, technology, and nutrition. Chapter 1 covers water and its relation to food. This book covers classical as well as new information on the glycobiology of carbohydrates in Chapter 2 and amino acids, oligopeptides, polypeptides, and proteins in Chapter 3. Chapter 4 covers new and classical applications of enzymes in the food industry. Lipids, fats, and oils are covered in Chapter 5, along with the latest developments. Nucleic acid and DNA identification of foods are covered in Chapter 6, along with other applications. Chapters 2 through 6 include the most recent developments along with their applications in food and nutrition. There are several topics included in these chapters that are not covered in most food chemistry textbooks. The essential topic of plant, animal, and human hormones and their risks and benefits to producers and consumers are covered in Chapter 7. Chapter 8 covers the topic of functional foods, herbs, and dietary supplements. Most classical food chemistry/biochemistry books mention close to 20 carbohydrates (glucose, fructose, lactose, etc.), 22 basic amino acids, 20 or more fatty acids, 13 vitamins, and 30 minerals in food systems. But now, we know that foods may contain at least 100,000 different chemicals. Chapter 8 covers, classifies, and explains the functions of some these compounds. Herbs and dietary supplements are considered as food for all legal and practical purposes. The recent renewal of interest in herbal medicines has made it necessary to have a clearer understanding of their structure and function in human diet. Natural dietary supplements can include a variety of products derived from or by products of foods, can be high in certain ingredients, and can contain bioactive compounds that aid in maintaining the health and wellness of individuals. Chapter 9 covers flavor compounds in foods, while Chapter 10 deals with organic acids, including phenolic acids. Other topics covered include the interactions of the environment with food components, that is, oxidations and changes of foods during their life cycles. Chapters 11 and 12 cover biological and lipid oxidations in foods and their controls. Chapter 13 covers the issue of food safety—major contaminants and additives are discussed and their regulatory limits and status are explained. Chapters 14 and 15 summarize the interactions of nutrition and the genetic makeup of the individual components. Chapter 14 covers an emerging field of food research and focuses on identifying and understanding, at the molecular level, interaction between nutrients and other dietary bioactives with the human genome during transcription, translation and expression, the processes during which proteins encoded by the genome are produced and expressed. Chapter 15, which deals with toxicogenomics, discusses the interaction of food additives and contaminants with the genome of the individuals, and how this translates into human disease conditions. The rapid growth of the food and dietary supplement industry into big business and the increase in the number of items on the shelves, the new controls on food additives and contaminants, and

ix

x

Preface

the attempts to standardize these articles all serve to indicate the growing importance of this timely book on advances in the biochemistry of foods. Thus, the aim of this book is to provide a unified picture of foods from a biochemical point of view. The primary emphasis is on the technological, nutritional, health functionality, and toxicological properties of foods, and the changes that occur during the processing, storage, preparation, and consumption processes. The authors have written this book trying to keep in mind undergraduates and graduates who have had several courses in chemistry/biology; however, an effort has been made to provide the necessary background in chemistry/biology and sufficient explanations/illustrations for students to proceed without additional references. I would like to thank the authors, reviewers, publishers, and all those involved in making this book a reality. Fatih Yildiz Middle East Technical University Ankara, Turkey

Editor Dr. Fatih Yildiz received his BS from Atatürk University in Erzurum, Turkey. He received his MS and PhD in food biochemistry from the University of Maryland, College Park. He received his assistant and associate professorships from the Middle East Technical University, Ankara, Turkey. He worked as a faculty member at the University of Maryland for five years. Dr. Yildiz worked as a professor in the Department of Food Science and Nutrition at the University of Minnesota, Minneapolis. He has also conducted research at the Institut National Recherches Agronomiques (INRA), Avignon, France as a visiting professor in 1997. Currently, he teaches and conducts research in the biochemistry, biotechnology, and food engineering departments at the Middle East Technical University, Ankara, Turkey. He has worked on research projects with the Food and Agriculture Organization of the United Nations (FAO), United Nations Industrial Development Organization (UNIDO), the United Nations Children’s Fund (UNICEF), and the North Atlantic Treaty Organization (NATO) as a project director. Dr. Yildiz has published more than 130 research and review papers, mostly in English, in international and national journals as the major author. He has published in Turkish, French, and German. His research studies have been cited by Science Citation Index (SCI) more than 50 times. He has coauthored a book, Minimally Processed and Refrigerated Fruits and Vegetables, published by Chapman & Hall in 1994, which was then a new concept in the food industry. His current research interests include health nutrition and safety attributes of Mediterranean diet. He has edited a book, Phytoestrogens in Functional Foods, published by CRC Press. Professor Yildiz, is listed in the Who’s Who in Turkey and Europe and serves on numerous advisory committees of the Ministry of Health and Agriculture. He is also a member of the National Codex Commission in Turkey, and a member of 10 scientific and academic organizations in the United States, France, and Turkey. Scientific and professional society memberships that Professor Yildiz is a member of 1. 2. 3. 4.

Institute of Food Technologists, Chicago, IL (since 2001) Turkish Food Technologist Association, Ankara, Turkey (since 1981) American Society for Microbiology, Washington, DC (since 1975) Global Food Traceability Forum, Halifax, West Yorkshire, United Kingdom (member since 2004) 5. European Federation of Food Science and Technology, Wageningen University, Wageningen, the Netherlands 6. Who’s Who in Turkey and Who’s Who in European Research and Development

xi

xii

Editor

Honors and awards 1. INRA Research Award (1997) on Post-Harvest Mushroom Technology, INRA, Avignon Research Station, France 2. Minnesota–South Dakota Dairy Foods Research Center Grant to conduct research in the University of Minnesota, Minneapolis (1991–1992) 3. Gaziantep Chamber of Commerce and Industry Award for outstanding service to the region (1998) 4. Ambassador for Turkey Award (2008) by EFFoST

Contributors Dimitris G. Arapoglou Laboratory of Biotechnology National Agricultural Research Foundation Athens, Greece Ioannis S. Arvanitoyannis School of Agricultural Sciences, Animal Production and Agricultural Environment University of Thessaly Volos, Greece Bruno Biavati Department of Agroenvironmental Science and Technology University of Bologna Bologna, Italy Sara Bosi Department of Agroenvironmental Science and Technology University of Bologna Bologna, Italy Dilek Boyacioglu Department of Food Engineering School of Chemical and Metallurgical Engineering Istanbul Technical University Istanbul, Turkey Esra Capanoglu Department of Food Engineering School of Chemical and Metallurgical Engineering Istanbul Technical University Istanbul, Turkey Pietro Catizone Department of Agroenvironmental Science and Technology University of Bologna Bologna, Italy

Muharrem Certel Faculty of Engineering Department of Food Engineering Akdeniz University Antalya, Turkey Çag˘atay Ceylan Department of Food Engineering Izmir Institute of Technology Urla, Turkey Meltem Yalinay Cirak Faculty of Medicine Department of Microbiology and Clinical Microbiology Gazi University Ankara, Turkey Giovanni Dinelli Department of Agroenvironmental Science and Technology University of Bologna Bologna, Italy Abdullah Ekmekci Faculty of Medicine Department of Medical Biology and Genetics Gazi University Ankara, Turkey Sibel Fadılog˘lu Department of Food Engineering University of Gaziantep Gaziantep, Turkey Diana Di Gioia Department of Agroenvironmental Science and Technology University of Bologna Bologna, Italy

xiii

xiv

Fahrettin Gög˘üs¸ Department of Food Engineering University of Gaziantep Gaziantep, Turkey Bensu Karahalil Faculty of Pharmacy Department of Toxicology Gazi University Ankara, Turkey Barçın Karakas¸ Faculty of Engineering Department of Food Engineering Akdeniz University Antalya, Turkey Ayhan Karakoç Department of Endocrinology Gazi University Ankara, Turkey Sotirios Kiokias Laboratory of Food Chemistry and Technology School of Chemical Engineering National Technical University of Athens Athens, Greece Is¸ıl A. Kurnaz Faculty of Engineering and Architecture Genetics and Bioengineering Department Yeditepe University Istanbul, Turkey Athanasios E. Labropoulos Food Technology and Nutrition Department Technological Educational Institute of Athens Athens, Greece Frank A. Manthey Department of Plant Sciences North Dakota State University Fargo, North Dakota

Contributors

Ilaria Marotti Department of Agroenvironmental Science and Technology University of Bologna Bologna, Italy Dilara Nilufer Department of Food Engineering School of Chemical and Metallurgical Engineering Istanbul Technical University Istanbul, Turkey Çig˘dem Soysal Department of Food Engineering University of Gaziantep Gaziantep, Turkey Theodoros H. Varzakas Department of Food Technology School of Agriculture Technological Educational Institution of Kalamata Kalamata, Greece Y. Sedat Veliog˘lu Faculty of Engineering Department of Food Engineering Ankara University Ankara, Turkey Yingying Xu Department of Cereal and Food Sciences North Dakota State University Fargo, North Dakota Fatih Yildiz Department of Food Engineering and Biotechnology Middle East Technical University Ankara, Turkey

1 Water and Its Relation to Food Barçın Karakas¸ and Muharrem Certel CONTENTS 1.1 1.2

Abundance and Significance of Water in Foods.......................................................................1 Chemical and Physical Properties of Water .............................................................................2 1.2.1 Water Isotopes ..............................................................................................................4 1.2.2 Types of Water ..............................................................................................................4 1.3 Water as a Solvent .....................................................................................................................5 1.4 Concepts Related to Water Mobility in Food ...........................................................................8 1.5 Drinking Water ....................................................................................................................... 11 1.5.1 Water Contaminants ................................................................................................... 14 1.5.1.1 Microbiological Parameters ......................................................................... 15 1.5.1.2 Chemical Parameters ................................................................................... 16 1.5.1.3 Radionuclides ............................................................................................... 19 References ........................................................................................................................................ 19

1.1 ABUNDANCE AND SIGNIFICANCE OF WATER IN FOODS Water can universally be found in the solid, gaseous, and liquid states. Saltwater oceans contain about 96.5% of our global water supply. Ice, the solid form of water, is the most abundant form of freshwater and most of it, nearly 68.7%, is currently trapped in the polar ice caps and glaciers [1]. About 30% of the freshwater sources are present in aquifers as groundwater. The remaining freshwater is surface water in lakes and rivers, soils, wetlands, biota, and atmospheric water vapor. Climate change due to global warming coupled with the ever-increasing demand for water by a growing human population have a surmounting effect on the present and future scarcity of this valuable resource. Besides the unavailability of water itself, shortages also result in problems, such as increased impurities and contaminants in the available water. In facing these situations, populations are obligated to manage and reuse water sources with awareness and diligence. Water is the most abundant and surely the most frequently overlooked component in foods. It is estimated that over 35% of our total water intake comes from the moisture in the foods we consume [2]. The other contributors to our water intake are beverages and metabolic water, which is produced through chemical reactions in the body. The water content of foods is very variable. It may be as low as 0% in vegetable oils and as high as 99% in some vegetables and fruit. Water by itself is free of calories and plain water does not contain nutritive substances, but it may be an ingredient itself in foods. Foods are described as dry or low-moisture foods if they have very low water content. These are most often solid food systems. Liquid food systems and tissue foods where water is the dominating constituent of the solution are high-moisture foods. Foods that contain moderate levels of water are intermediate-moisture foods (IMFs). For the food industry, water is essential for processing, as a heating or a cooling medium. It may be employed in processes in the form of liquid water or in the other states of water such as ice or steam. 1

2

Advances in Food Biochemistry

Almost all food-processing techniques involve the use or the modification of water in food. Freezing, drying, concentration, and emulsification processes all involve changes in the water fraction of the food. Without the presence of water, it would not be possible to achieve the physicochemical changes that occur during cooking such as the gelatinization of starch. Water is important as a solvent for dissolving small molecules to form solutions and as a dispersing medium for dispersing larger molecules to form colloidal solutions. Water has historically been the primary solvent of choice for the extraction of apolar substances. Recently, the use of subcritical water, hot water at 100°C–374°C under high pressure, is being investigated as an environmentally friendly alternative to organic substances that are used for the extraction of polar substances [3]. The control of water activity in foods is an important tool for extending shelf life. It is responsible for the quality of foods affected by microbiological, chemical, and physical changes. The physical properties, quantity, and quality of water within the food have a strong impact on food effectiveness, quality attributes, shelf life, textural properties, and processing. Food-preservation processes have a common goal of extending the shelf life of foods to allow for storage and convenient distribution. The activity of microorganisms is the first and most dangerous limitation of shelf life. Water is essential for microorganisms that may cause food spoilage if they are present in a food that offers them favorable conditions for growth. Hence, many foodpreservation techniques were developed to reduce the availability or activity of water in order to eliminate the danger of microbial spoilage. The presence or activity of water in foods may also enhance the rate at which deteriorative chemical reactions occur. Some products may become rancid through free radical oxidation even at low humidities and thus become unacceptable. Labile nutrients such as vitamins and natural color compounds are oxidized more rapidly when stored at low moisture levels. Enzyme-mediated hydrolytic reactions may reduce the quality of the food product. Other reactions such as the Maillard type of nonenzymatic browning may be enhanced by the presence of higher levels of water. On the other hand, water content is crucial for the textural characteristics and the sensory perception of foods. A food may be found unacceptable by consumers simply because it does not satisfy their textural (sensory) anticipation. For the reasons mentioned above, it is important to have control over the quantity and quality of water in foods and in the processing thereof. In order to do so, it is essential to have a better understanding of the chemical and physical properties that determine the fundamental functions of water. Controlling the growth of ice crystals is a primary concern for food technologists. Developments in the field of molecular biology have enabled recombinant production of many proteins. Two of these protein groups are the antifreeze proteins [4] and ice nucleation proteins [5,6], which have potential applications in the food industry. The application of these proteins and other techniques such as dehydro freezing, high-pressure freezing/thawing, and ohmic and microwave thawing are other options that are now available for achieving rapid freezing or thawing of foods [7].

1.2 CHEMICAL AND PHYSICAL PROPERTIES OF WATER Water, with the chemical formula H2O, has unique properties determining its physical and chemical nature. Water behaves unlike other compounds of similar molecular weight and atomic composition, which are mostly gasses at room temperature. Some physical properties of water are presented in Table 1.1. It has relatively low melting and boiling points, unusually high values for surface tension, permittivity (dielectric constant), and heat capacities of phase transition (heat of fusion, vaporization, and sublimation). Another unusual behavior of water is its expansion upon solidification. The unexpected and surprising properties of water can be better understood after taking a closer look at its structure at a molecular level. The inter- and intramolecular forces involved are governed by the physical and chemical states and reactions of water.

Water and Its Relation to Food

3

TABLE 1.1 Some Physical Properties of H2O Property Freezing temperature at 101.3 kPa (1 atm) Boiling temperature at 101.3 kPa (1 atm) Heat of fusion at 0°C and 1 atm Heat of vaporization at 100°C and 1 atm Heat capacity of water at 20°C Heat capacity of ice at 0°C Density of water at 20°C Density of ice at 0°C Dielectric constant of water at 20°C

Value 0°C 100°C 6.01 kJ/mol 40.66 kJ/mol 75.33 J/mol K 37.85 J/mol K 0.998 g/mL 0.917 g/mL 80.4

A molecule of water consists of two hydrogen atoms that are each bonded to an oxygen atom by a covalent bond, which is partially ionic in character. In this conformation where oxygen is at the center, there is an angle of 104.5° between the nuclei. Oxygen is strongly electronegative and can be visualized as partially drawing away the electrons from the hydrogen atoms leaving them with a partial positive charge. Due to the partially ionic nature of the bonds, the spatial localization of the electrons forms a tetrahedral quadruple where the oxygen is at the center and the two negative charges of oxygen’s lone pair electron orbitals and the two positive charges of the hydrogen atoms form the four corners. The structure is polar, and due to this separation of charges the molecule behaves like an “electric dipole.” This polarity represents the governing force between the water molecules rather than the weaker van der Waals forces. The water molecule is attracted to neighboring water molecules by the affinity of the positive charged site of one molecule to a negative charged site of its neighbor and vice versa. This attractive force is termed as a “hydrogen bond.” Each water molecule can therefore support four hydrogen bonds. Hydrogen bonds are stronger than that of van der Waals’ and much weaker than covalent bonds, but the ability of a water molecule to form multiple hydrogen bonds with its neighbors in three dimensional space can help to visualize the effectiveness of these forces in establishing the strong association of water molecules. This strong association between the water molecules provides a logical explanation for its unusual properties. Similar interactions occur between the OH and NH groups and between strongly electronegative atoms such as O and N. This is the reason for the strong association between alcohol, amino acids, and amines and their great affinity to water. Intra- and intermolecular hydrogen bonding occurs extensively in biological molecules. A large number of the hydrogen bonds and their directionality confer very precise three-dimensional structures upon the proteins and nucleic acids. Water may influence the conformation of macromolecules if it has an effect on any of the noncovalent bonds that stabilize the conformation of the large molecule. These noncovalent bonds may be hydrogen bonds, ionic bonds or apolar bonds. The arrangement of water molecules in ice is very close to a perfect tetrahedral configuration. The angle formed between the oxygen atoms of three adjacent water molecules is approximately 109°. The same angle for liquid water is about 105°. Because liquid water has a smaller bond angle than ice, molecules in water can be packed more closely and so water has a greater coordination number, which is basically the average number of the neighbors. Ice is therefore less dense and has a lower density than liquid water. Liquid water has the greatest density at about 4°C. Above this temperature as heat is introduced into the system, the hydrogen bonds weaken and the intramolecular space increases.

4


Although, for the sake of visualization, the explanation made above pictures a static molecular and intermolecular conformation of water and ice, in reality the atoms in the molecules and the molecules themselves are highly dynamic. In water the hydrogen bonds break and reform with high frequency. Even in ice, where the molecules are bound with stronger forces, water molecules are thought to vibrate and diffuse in the interstitial spaces within the crystal lattice. Another point to be considered is the dissociation of water molecules to hydronium (H3O+) and hydroxyl ions (OH−) that complicates the structural formations by providing altered potentials for the hydrogen bond formation. 2H 2O ↔ H 3O + + OH − Water ionizes reversibly as shown in the above equation and the dissociation reaction occurs in equilibrium. Therefore, in pure water, the number of ionized water molecules at any given time is the same. The concentration of the dissociated molecules is equal to the concentration of the hydronium ions, 10 −7 mol/L that is equivalent to a pH of 7.

1.2.1

WATER ISOTOPES

Oxygen is known to exist in nature as six stable isotopes (14O, 15O, 16O, 17O, 18O, and 19O). Similarly, there are three known isotopes of hydrogen (1H, 2H (deuterium, D), and 3H (tritium)). Of these isotopes, 14O, 15O, 19O, and tritium are radioactive with relatively short half-lives and their presence in natural water is not significant. Water consists of the stable isotopes and the majority of water will consist of H216O. The abundance of the other stable isotopes may vary depending on the origin of the water but within the limits of variation, the abundances of the remaining isotopes H218O, H217O, and HDO may be stated as 0.2%, 0.04%, and 0.03%, respectively. Isotopes share the same chemical properties because they depend only on the number of protons. But the difference in the number of neutrons will result in different weights and this is particularly evident in the case of hydrogen. Deuterium is twice as heavy as 1H. Isotopes of an element can have different weights and this will cause different physical properties. Due to its extra weight deuterium heavy water (D2O) boils at 101.4°C and freezes at 3.8°C. The difference in weight affects the speed of the reactions involving water. For this reason, heavy water is not safe to drink [8]. Since the preparation of pure H216O is extremely difficult, virtually all existing experimental determinations on water were performed on the naturally occurring substance. Techniques involving the use of D2O and nuclear magnetic resonance (NMR) imaging were developed as noninvasive methods to observe the time course of water transport in microporous food materials [9]. The assessment of the stable isotope ratio of foods is a convenient method that was developed for the determination of fraud in the food products industry. Determination of D and 18O content is a method utilized in the determination of watering of fruit juices and wines [10]. Stable isotope ratios of 13C/12C, 15N/14N, 18O/16O, and 2H/1H are useful in the discrimination of other adulteration schemes and mislabeling of food products [11,12]. Enzymes may be sensitive to the presence of heavy isotopes and the isotope selection effects may be high for certain transformations, especially for hydrogen. The D/H ratio can thus be used to determine the mechanism of the action of enzymes and biosynthetic pathways [13].

1.2.2

TYPES OF WATER

Water in foods and biological materials can be grouped in three categories: free water, entrapped water, and bound water. Free water is easily removed from foods or tissues by cutting, pressing, or centrifugation. Entrapped water is immobilized within the lattices of large molecules, capillaries,


5

or cells. Entrapped water, although not free flowing, does have the properties of free water. Free and entrapped water together may be considered bulk water. This water will behave almost like pure water during food processes. It is easily removed by drying, easily converted to ice during freezing, and available as a solvent. Bound water however, exhibits properties significantly altered than those of bulk water in the same system. Bound water is the layer of water molecules closest to solutes and other nonaqueous constituents and is more structurally bonded than free or entrapped water thus having hindered mobility. Bound water is not free to act as a solvent for additional solutes, can be frozen only at very low temperatures below the freezing point of water, is denser than water, and exhibits no vapor pressure. In a medium-moisture food only a minute fraction of water that is directly in the vicinity of the solutes is bound water [14]. The fact that water molecules in solutions and in food systems are in constant motion applies to bound water as well as free water meaning that there is a constant exchange of individual water molecules at binding locations [15]. Although the mechanism of attraction allows this motion it is not practically possible to remove all bound water from foods by dehydration. This water is also resistant to freezing, is not available for chemical reactions and does not serve as a solvent [16]. The water mobility and its relation to water retention in foods is an area invoking interest. The effect of water mobility on the rate of chemical reactions is multidimensional and cannot be reduced to a single physicochemical parameter [17]. Water states and displacements can be investigated with thermogravimetry (TG) either in its classical or in the Knudsen version (where standard pans are replaced with Knudsen cells) [18]. The analysis of water content in foods is one of the most frequently and routinely performed analyses of foods. Methods used in the analysis of water are outlined in Figure 1.1. The great variability in water contents of food materials requires that the results of other analyses of foods are to be reported on a dry weight basis. This is one of the main reasons for the routine determination of water content in the food laboratory. While there is ongoing research to achieve improvements in the methods of water analysis [19,20], recent interest is also forwarded to the economic aspects of water determination in foodstuffs [21].

1.3 WATER AS A SOLVENT Water is a good solvent for most biomolecules, which are generally charged or polar compounds. The form and function of biomolecules in an aqueous environment are governed by the chemistry of

Determination of water in foods

Direct methods

Physical methods

Dessication to achieve an equilibrium Distillation Oven drying Infrared, halogen, and microwave drying

FIGURE 1.1 395, 2001.)

Indirect methods

Chemical methods

Combined methods

Calcium carbide and calcium hydride methods Karl fischer titration

Evaporation and Karl Fischer titration Evaporation and diphosphorus pentoxide method

Densimetry, polarimetry, refractometry, and electrical property measurements Use of water activity tables NMR spectroscopy NIR spectroscopy MW spectroscopy

Methods for determining water content of foods. (Based on Isengard, H.-D., Food Control, 12,

6


their component atoms and the effect of water molecules surrounding them. The polar and cohesive properties of water are especially of importance for its solvent characteristics. The introduction of any substance to water results in altered properties for the substance and water itself. The degree and type of alteration is dependent on the molecular and electronic structure, ionization characteristics, size, and stereochemistry of the solute. The substance introduced into water, depending on these properties, will be dissolved, dispersed, or suspended. Water dissolves small molecules such as salts, sugars, or water-soluble vitamins to form “true solutions” which may either be ionic or molecular. When different chemical and biochemical materials are introduced into water, there is a rise in the boiling point and viscosity and a decrease in the freezing point and surface tension. Solubility increases with increasing temperature as the heat introduced reduces water–water hydrogen bond attractions and facilitates solute hydration. Ionic solutions are formed when the solute ionizes in water. The ions of the molecule separate in water and are surrounded or “hydrated” by the water molecules. Ionic molecules greatly influence the mobility of water molecules surrounding them and affect the colligative properties of solvent water. The degree to which the structure of bulk water is disrupted depends on the valence, size, and concentration of the ion in solution. In ice, the presence of ions interferes with intermolecular forces between water molecules and disrupts the crystal lattice structure. Hence the presence of salt decreases the melting point of water. The great ionizing potential of water can be attributed to its high dielectric constant unmatched by other liquids [23]. Electromagnetic fields at radio and microwave frequencies interacts with food constituents, resulting in internal heat generation due to the dipole rotation of free water and the conductive migration of charged molecules [16]. Other molecules, such as sugars or alcohols dissolve in water to form molecular solutions. These hydrophilic molecules are also hydrated by water molecules clinging to them by hydrogen bonding but the solute molecules stay intact. In solution glucose molecules have a strong stabilizing effect on the clusters of water molecules around them. This gelling effect is more pronounced in the polysaccharides [24]. The same effect is true of the fiber polysaccharides that are generally less soluble and the gums. The presence of OH groups on the carbon atoms and the compatible stereochemistry of monosaccharides render them highly soluble organic compounds. As for the oligosaccharides, the presence of some hydrophobic regions in the solute molecules affects their solubility. In polysaccharides, although the formation of the gel structure requires an aqueous environment, the gelling properties of the polysaccharides are determined primarily by polymer–polymer interactions [25]. Molecules that are too big to form true solutions can also be dispersed in water. Smaller molecules ranging in size of up to 100 nm are dispersed to form colloidal dispersions. Such molecules are generally organic products and polymers such as cellulose, pectin, starch, or proteins. Generally a two-phase colloid solution of relatively lower viscosity, where the solute is dispersed in a matrix of water is termed a sol. A gel is also a two-phase colloidal dispersion however in this case the solvent is dispersed in a solute matrix forming an elastic or semisolid. In a gel, the solute matrix does not necessarily have to constitute the greater mass portion of the total. “Water holding capacity” is a term that is frequently used to describe the ability of a matrix of molecules, usually macromolecules at low concentrations, to physically entrap large amounts of water. The stability of colloid solutions is particularly vulnerable to factors such as heating, freezing, and changes in pH. The conformation of macromolecules and stability of colloids are greatly affected by the kinds and concentrations of ions present in the medium. Physical gels are formed when water is added to a lyophilic polymer but in insufficient amounts to completely dissolve the individual chains. Various polysaccharides such as pectin, carrageenan, and agarose, and proteins such as gelatin form physical gels in aqueous solution. These types of gels are usually reversible, i.e., they can be formed and disrupted by changing the pH, temperature, and other solvent properties.


7

When particles too large to form colloidal solutions, greater than 100 nm are dispersed in water they form suspensions. When the dispersion involves nonsoluble (nonpolar) liquids this is called an emulsion. Emulsions of water and oil may consist of droplets of oil in water (o/w emulsion) or droplets of water in oil (w/o emulsion) depending on which is the dispersed and continuous phase. Suspensions and emulsions have a tendency to separate when left to stand for long periods of time. Addition of stabilizers or emulsifiers to these solutions may facilitate dispersion and protect the solution from phase separation. Some foods, like margarine, butter, and chocolate, consist of semisolid fat in a continuous phase. In these foods, formation of water bridges results in an increase in the yield stress value of semisolid fat upon the addition of water [26]. Water interacts strongly with polar groups and resists nonpolar structures, such as molecules containing carbon atom chains. In an aqueous environment, there is a tendency for water to be more ordered, forming lattice-like structures around these nonpolar molecules. Hence, there is a tendency for the nonpolar molecules to aggregate and release some of the water molecules, increasing the total entropy of the water molecules. This phenomenon is called the hydrophobic effect or hydrophobic interaction. The hydrophobic effect plays an important role in many biochemical processes. Most biomolecules are amphiphilic, meaning they have both hydrophilic and hydrophobic groups. In an aqueous environment, these molecules self-assemble where the apolar parts of the molecules are grouped together. Some examples of structures formed as a result of this self-assembly are illustrated in Figure 1.2. The lipid bilayer is in fact the backbone of all biological membranes that makes compartmentalization possible in all cells. The assembly of these molecules would not be possible without the presence of water around them. Protein–water interactions are vitally important in the application of proteins in model and food systems. Physicochemical properties of proteins such as dispersibility and viscosity are directly affected by solubility. Water associates with proteins in a progressive manner that may range from water molecules associated with specific groups to the more hydrodynamic hydration layers. The functional properties of a protein are determined by its three dimensional conformation determined by the water molecules surrounding it [27,28]. Natural proteins are stable globular structures consisting of carbon rich amino acids that form the core, taking advantage of the hydrophobic effect. The charged and hydrogen bonding amino acids that tend to associate with water to give the protein its specific stereochemistry, tend to position themselves at the globule interface. In this dynamic equilibrium state, charged groups of the protein are almost always located at the surface of the native protein whereas the interior contains hydrophobic groups tucked away from the surrounding water molecules (Figure 1.3). When the protein is denatured it loses functionality and the coil expands as all groups are hydrated and the charge distribution becomes more even [29]. Water

Water

Oil

Water (a)

(b)

(c)

FIGURE 1.2 Schematic representation of structures formed as a result of lipid assembly in solution in biological and food systems. (a) Cross sections of a lipid bilayer, (b) a micelle, and (c) an inverted micelle.

8


r΄ r

Native protein

Denatured protein

FIGURE 1.3 Schematic representation of protein conformation in an aqueous environment. (Adapted from Norde, W., Colloids and Interfaces in Life Science, Marcel Dekker Inc., New York, 2003, 1–433.)

1.4 CONCEPTS RELATED TO WATER MOBILITY IN FOOD Biochemical constituents may partially immobilize water by stopping vaporization and lowering its chemical reactivity. The state and availability of water in food is just as important as the content of water. As a measurable quantity, water activity, expressed as aw is used to express the availability of water in a food product. It is defined as the partial lowering in partial pressure created by the food and is the ratio of the partial pressure of water in the food to the partial pressure of pure water at the same temperature. Water activity is also equal to the equilibrium relative humidity (ERH) divided by 100. ⎡p ⎤ aw = ⎢ food ⎥ = % ERH ⎣ pwater ⎦ P ,T Water activity in all foods is always a value below 1.0 because water is associated with the surfaces and solutes in the food. Differences among water activities of food components, food domains, and the external environment outside the food induces a driving force of water transport from high aw to low aw until a common equilibrium value for the food system is reached. Moisture sorption isotherm is a graphical representation of the variation in water activity or % ERH with change in moisture content of a sample at a specified temperature. Sorption isotherms of foods are generally nonlinear and very often sigmoid in shape (see Figure 1.4). The difference in the equilibrium water content between the adsorption and desorption curves is called hysteresis. The hysteresis phenomenon is observed in highly hygroscopic materials [30]. The curve is divided into three regions. In the first region, the water in the food consists of water that is tightly bound to the product. In the second region, there is more water available and it is less tightly bound and present in small capillaries. In the third region, where the food contains higher levels of water and has a high aw, there is plenty of water that is free or loosely held in capillaries. A detailed knowledge of water sorption isotherms are essential in food technology applications such as concentration, dehydration, and drying of foods, as well as in determining the quality, stability, and shelf life of foods. Statistical models of the sorption behavior of foods have been applied for predicting the sorption behavior of foods. One of the most well-known and used model is the equation proposed by Brunauer, Emmett, and Teller known as the BET sorption isotherm model. This model is used extensively in food research. Another model extensively used for foods proposed by Guggenheim, Anderson, and de Boer is known as the GAB model or equation. The predicted values by either


9

Equilibrium water content (% dry basis)

Bound water

Bulk water

Desorption

Adsorption

0

FIGURE 1.4

Entrapped water

%ERH

100

Sorption isotherm for a food product showing hysteresis.

equation are equivalent to the critical minimum moisture levels in foods above which sensory properties change and rehydration occurs. The storage life of foods is influenced significantly by the water activity in the product. Intermediate-moisture foods, which have aw values between 0.6 and 0.9, have drawn considerable attention [31]. Many of the intermediate-moisture foods have reduced moisture contents and are palatable without the need to rehydrate them [32]. Microbial proliferation is terminated at aw values below 0.6. Even at higher moisture levels, the effect of lowered aw has a synergistic effect in prolonging shelf life when combined with other methods of preservation. This technique of combining multiple preservation techniques to extend the shelf life stability of foods is known as the hurdle technology. aw and temperature are important factors influencing the state of amorphous food materials that are lacking in molecular order. These materials can be either in the rubbery or the glassy state. In the case of the latter, the material can be described as rigid, yet brittle, and has a high internal viscosity and a high internal mobility. The same amorphous material will become rubbery if the temperature is increased or if water is added into the system as a plasticizer. The temperature at which the critical conversion takes place is the glass transition temperature (Tg). Glass transition is a kinetic equilibrium process at temperatures below Tg. Mapping of food stability in relation to aw and Tg is for the moment not extensive, however, a better understanding of the sorption properties of foods and Tg can be used in controlling stability and food packaging requirements [33]. The glass transition allows for the prediction of several physical evolutions in food products at low water content or in the frozen state which affect stability. The glass transition temperature is of importance especially for dry food products that are predominantly in a glassy amorphous form. When the products are stored at temperatures above Tg, the increased rate of physicochemical reactions that take place causes sticking, collapse, caking, agglomeration, crystallization, loss of volatiles, browning, and oxidation, which ultimately result in quality loss [34]. It is increasingly recognized that other parameters besides Tg, such as fragility, αβ crossover temperature, and distribution of relaxation times, can also be used to study the time- and temperature-dependent distribution and mobility of water in foods [35]. The state diagram shown in Figure 1.5 is a plot of the states of a food as a function of the water or solids content and temperature. Most of the transitions of state can be measured by the differential scanning colorimetry (DSC) method, which detects the change in heat capacity occurring over the glass transition temperature range. Mechanical spectroscopy (or dynamic mechanical thermal

10

Advances in Food Biochemistry Liquid

Solid

Water vapor

P D Crystal Collapse Stickiness Rubber Softening zone Reaction zone N C Entangle zone

M A Solution H

Tu I

Tm

J G

Tg΄

S

Tgw

Ice + solution

B

Ice + solution + solute Ice + rubber + solute Ice + glass + solute Ice + glass + solute

Q

F

Glass

R

X s΄

0

TMS

Tgs E BET-monolayer

Temperature

Tbw

L

O

1

Solute mass fraction

FIGURE 1.5 State diagram of foods (Tbw, boiling point; Tu, eutectic point; T m′ , end of freezing; T g′ , glass transition at endpoint of freezing; Tgw, glass transition of water; Tms, melting point of dry solids; Tgs, glass transition of dry solids. (From Rahman, M.S., Trends Food Sci. Technol., 17, 129, 2006. With permission.)

analysis, DMTS) and dielectric spectroscopy are alternatives, and some times more sensitive methods aimed at determining the change in the state of the food [35,36]. Physical and structural changes affecting quality of foods, such as collapse during drying, stickiness, and caking of dry powders, crystallization, change in viscosity or other textural changes, occur principally when the storage or processing temperature is above Tg. The rate of chemical reactions taking place in foods can be described using the Arrhenius relationship: k = k0 exp (− Ea RT ) In this model, the rate constant, k, is expressed as a function of the pre-exponential factor, k0, the ideal gas constant, R, temperature, T, and the activation energy, Ea. However, the Arrhenius temperature model often falls short of explaining the physical behavior of foods, especially of macromolecular solutions at the temperatures above Tg. A better description of the physical properties is offered by the Williams–Landel–Ferry (WLF) model, which is an expression relating the change of the property to the T − Tg difference [37,38]. That is, log aT =

−C1 (T − Tg ) C2 + T − Tg

where aT is the ratio of the relaxation phenomenon (i.e., η/ηg for viscosity) C1 and C2 are system-dependent constants As a plasticizer or softener for macromolecular food structures, water reduces Tg due to the reduction in the inter- and intramolecular forces. Freezing, drying, and increasing the content of solutes in a food system are practical methods of decreasing aw and increasing shelf life. The Tg curve can be used to determine the critical value for water activity (“critical aw”) equivalent to the “bound” or “monolayer” water activity value. Plots


11 0.25

30 0.2 20 0.15

10 0

0.1

–10 0.05

Moisture content (fraction)

Glass transition temperature, Tg (°C)

40

–20 0

–30 0

0.2 0.4 0.6 0.8 Water activity, aw (fraction)

1

FIGURE 1.6 Use of the sorption isotherm and state diagram in determining the critical aw. (From Sablani, S.S. et al., Int. J. Food Prop., 10, 61, 2007. With permission.)

combining the glass transition and sorption curves as shown in Figure 1.6 can be useful in identifying the critical moisture content of a food at a certain processing or storage temperature.

1.5 DRINKING WATER The individual requirement for the consumption of fluids may differ greatly depending on the physical activity and weight of the person as well as the ambient conditions. Nevertheless, a person is recommended to consume the equivalent of 1 mL/kcal energy expenditure [40] or at least 2–3 L of water daily, apart from the water ingested with foods and beverages. A potable water supply is essential for the survival of human life. Society, in general, requires water for the maintenance of public health, fi re protection, cooling, electricity generation, use in industrial and agricultural processes, and navigation. Within the concept of food, water may be utilized for different purposes: direct consumption as drinking water, as a primary source during agricultural production of plant and animal products, and during the industrial and domestic processing of foods. It is not a requirement for water to be extra pure in order for it to be potable. In fact many of the minerals necessary for our health are present as solutes in water. The presence of solutes in water not only contributes to the beneficial presence of minerals, but also to the desirable pleasant taste of the water. On the other hand water may be contaminated by chemical, physical, microbiological, or radiological factors of natural or anthropogenic origin. The levels and types of various contaminants actually form the basis for the definition of the quality of water. The adequacy of water for specific uses may be decided on by its quality. The quality of water, as true for other commodities, depends on its origin or source, the factors which influence its composition during transport and storage, and on the processes which have been applied to improve its quality. The concerns for quality can, therefore, be source related if the source of water is contaminated, treatment related if the concern stems from the techniques used to treat water, or distribution related if it is the distribution system that is in question. The quality requirements for water depend on its intended use, and the criteria identified for monitoring quality are determined accordingly.

12


Potable drinking water can be defined as the water delivered to the consumer that can be safely used for drinking, cooking, and washing. Drinking water is intended for direct consumption for humans, and, thus, must be free of any hazards to health. Providing wholesome, clean, and safe water to the masses requires control that can only be achieved by setting standards, monitoring of quality parameters, and legislative directives which must be adhered to. Drinking water supplied to the consumer must meet the physical, chemical, microbial, and radionuclide parameters or standards. Analysis and periodic monitoring are primary to achieve quality standards. However, a more total quality approach is required [39]. Periodic control and review of water treatment and transport facilities, evaluation and protection of the water source, and the evaluation of the quality and performance of the laboratory analyses are also essential to achieve better quality. Implementation of the hazard analysis critical control points (HACCP) approach to water supplies and use, specifically in the food industry, has been recommended [41,42]. The hazards encountered in water treatment in food factories must be identified and controlled. Possible hazards encountered have recently been presented as a report of the European Hygienic Engineering and Design Group [43]. The setting of standard limits and practicability of the water monitoring system of control depends on the identification and quantification or analysis of the contaminants present. Recent advances in the species of contaminants and the techniques used for their analyses have made it possible to better standardize and regulate water quality. Freshwater sources generally consist of ground and surface water sources. In rare cases, water may be obtained from sea or ocean water by desalination processes that are relatively costly. Most municipal systems utilize surface water whereas most of the industrial consumers of water prefer to pump water from ground sources than to obtain it from municipal systems. Tap water provided by the municipal system to the community is treated to an extent which generally finds a balance between the economy and the practicality of the treatment and the safety of the water delivered. Nevertheless, the basic principle is that the quality of the water should be suitable for consumers to drink and use for domestic purposes without subsequent risk of adverse effects on their health throughout their lifetime. Also, special attention is necessary to protect vulnerable groups, such as pregnant women and children. Agencies, international, state, or local, involved in the establishment of quality control of water have the common primary aim of protecting public health. Internet addresses of the U.S., the EU, and the WHO organizations concerned with water quality are presented in Table 1.2. There are international agencies such as the World Health Organization (WHO) that set guidelines for the quality of drinking water [44,45]. Much of the WHO work is associated with water supply and sanitation in developing countries. The evaluation of risk, extrapolation of data, and the acceptance of risk are used to develop guidelines on drinking water quality. Many of these guidelines have subsequently been included in the EU directives and the legislation of other

TABLE 1.2 Internet Addresses where More Information on Standards, Guidelines, and Legislation Can Be Obtained Area of Application

Organization

International

WHO

United States

USEPA

EU

Council of the EU

Address http://www.who.int/water_sanitation_ health/dwq/en/ http://www.epa.gov/safewater/creg. html http://ec.europa.eu/environment/water/ water-drink/index_en.html


13

governments in monitoring and sustaining drinking water quality. These WHO guidelines are applicable to packaged water and ice intended for human consumption. The regulations determining water quality differ from state to state and from country to country; however, generally, it is the environmental agencies that are responsible for regulating the quality of municipal water as well as providing the legal grounds for the protection of freshwater sources from environmental pollution. In the United States, the Safe Drinking Water Act (SDWA) authorizes the Environmental Protection Agency (EPA) to establish national health-based standards that reduce public exposure to the contaminants of concern. Each contaminant of concern has an unenforceable health goal, also called a maximum contaminant level goal (MCLG), and an enforceable limit referred to as maximum contaminant level (MCL). The EPA promulgates the National Primary Drinking Water Regulations (NPDWRs) that specify enforceable MCLs or treatment techniques for drinking water contaminants. The NPDWRs contain specific criteria and procedures, including the requirements for the monitoring, analysis, and quality control so that the drinking water system is in compliance with the MCL. The EPA sets MCLs as close to MCLGs as is technically and economically feasible. When the hazard of concern cannot be analyzed adequately, the EPA can require for a certain treatment technique to be employed during processing instead of setting a MCL. The EPA also sets secondary drinking water regulations that set monitoring recommendations or secondary MCLs for contaminants that affect the aesthetic, cosmetic, or technical qualities of drinking water. The water policy of the EU is guided by directives set by the EU, which member states adopt by enacting laws in accordance with these directives. The management of water as a resource is controlled by the water framework directive (2000/60/EC), and the control of discharge of municipal and industrial wastewater is controlled by the urban waste water treatment directive (91/271/EEC). Drinking water in the EU is comprehensively regulated by the current drinking water directive 98/83/EC adopted in 1998. This directive is currently under revision for improvement. Monitoring and compliance to essential quality and health parameters are obligatory for the member states by the EU Drinking Water Directives (DWDs) [46]. The DWDs cover all water intended for human consumption except natural mineral waters, medicinal waters, and water used in the food industry not affecting the final product. Bottled waters are alternatives to drinking water from the tap. The term “bottled water” is a generic term that describes all water sold in containers. The consumption of bottled water has risen phenomenally over the past years and the bottled water industry has grown to accommodate the demand [47]. Bottled waters are classified as a food material. They are monitored by the national food agencies and some international institutions. Many countries require the manufacturer to indicate the source of the water and the date of production on the label. The International Bottled Water Association (IBWA) also inspects its members annually to check for compliance to the IBWA standards. In the United States, bottled waters are regulated by the Food and Drug Administration (FDA) and state governments. The laws and regulations prompting compliance are constantly under revision and development. With the advances in analytical techniques and better scientific knowledge of the impurities that may be present in water and their health implications necessitates modification of the current status. There is growing interest in the concept of a total quality approach toward water quality management to achieve a more comprehensive and preventive system of control rather than a system that relies on the compliance monitoring of treated waters [48]. The concept of risk assessment and risk management during the production and distribution of drinking water was introduced by WHO in the Guidelines for Drinking Water Quality in 2004. The methodology is considered for adoption by the European Commission as well as individual state governments. Water, unless highly purified by distillation or membrane filtration technologies, is destined to contain differing amounts of various solutes such as minerals or suspended particles. Pure water at 25°C has a pH of 7 and it is a very strong solvent. Raindrops dissolve atmospheric gasses as they are being formed and collect dust particles and colloidal material in their fall. Carbon dioxide

14


will readily dissolve in water and dissociate to form carbonic acid, reducing the pH. The ultimate source of water, meteoritic water, is thus acidic in nature. As the water accumulates in aquifers and open bodies of water, it picks up solutes that are present in the surrounding soil, sediment layers, and rocks. Calcium and magnesium dissolved in water are the two most common minerals that make water “hard.” The degree of hardness becomes greater as the calcium and magnesium content increases and is related to the concentration of multivalent cations dissolved in the water. Hard water may be a nuisance because it reacts with soap to produce soap curd and interferes with the cleaning processes. Where hot water is used the minerals in hard water may settle out of solution forming scales in pipes and equipment reducing efficiency and increasing the cost of maintenance. However, it has not been shown to be a health hazard. In fact, the National Research Council (National Academy of Sciences) states that hard drinking water generally contributes a small amount toward total calcium and magnesium in human dietary needs. They further state that in some instances, where dissolved calcium and magnesium are very high, water could be a major contributor of calcium and magnesium to the diet. Water is not a strong contributor of dietary magnesium however the form of magnesium in water is thought to offer higher bioavailability than magnesium in foods. Epidemiological studies suggest that a negative correlation exists between the hardness of drinking water and cardiovascular mortality. Magnesium deficiency accelerates the development of atherosclerosis and the induction of thrombocyte aggregation, and, consequently, it is described as a risk factor for acute myocardial infarction and for cerebrovascular disease [49]. Because of this, magnesium supplementation of drinking water has been suggested. Another mineral which is considered to be supplemented in drinking water is fluoride. Fluoride is added to drinking water to prevent the incidence of dental caries, especially seen in children with a fluoride-deficient diet. The subject is, however, a controversial one since excess fluoride is believed to have adverse effects such as dental fluorosis, which is the yellow staining of the teeth. Mineral water is water from an underground source that contains at least 250 ppm of total soluble solids. Minerals and trace elements dissolved in it must come from the source and cannot be added later. Some mineral waters are naturally carbonated at the source, however most brands supplying fizzy mineral water add the CO2 gas artificially. With the exception of a few minerals which increase the health potential of drinking water, the presence of all other constituents is considered to be contaminants that pose potential health hazards.

1.5.1

WATER CONTAMINANTS

Contaminants polluting our water may be chemical or microbiological. Chemical hazards are caused by the chemical compounds which may be inorganic or organic. They may be present in the water due to the natural source, such as arsenic, which is a natural component of some soils and may be dissolved in groundwater. Alternatively, the contaminant may have been introduced into the ecosystem or the water source by human activity, such as pesticides, that trickle down sediment layers and streams and find their way into our freshwater. Some contaminants are by-products of chemicals used during the disinfection processes applied. The types of chemical contaminants that are present in water are numerous. The chemical species emerging as contaminants is ever increasing. It is estimated that 1000 new chemicals are identified in water each year. Setting standards for and monitoring such a wide array of chemicals are impractical. Acute health problems are seldom associated with toxic chemical contaminants in water except on rare cases of massive accidents where a chemical may be introduced into the water supply in very large amounts. This type of contamination is also rarely dangerous, because often the toxic chemical makes the water unsuitable for consumption due to unacceptable taste, odor, or color. Microbial contaminants, on the other hand, have acute and widespread effects, and thus require a higher priority.


15

1.5.1.1 Microbiological Parameters Infectious diseases caused by pathogenic bacteria, viruses, and protozoa or by parasites are the most common and widespread health risk associated with drinking water. Diseases such as cholera, typhoid fever, dysentery, diarrhea, enteritis, and infectious hepatitis are transmitted primarily through human and animal excreta. Human and animal wastes from sewage and farmyard runoff are principal sources for microbiological pollution. A list of the most important microbial pathogens in water is given in Table 1.3. Other pathogens which are not listed also exist. Bacteria such as Pseudomonas

TABLE 1.3 Orally Transmitted Waterborne Pathogens and Their Significance in Water Supplies Pathogen

Health Significance

Burkholderia pseudomallei Campylobacter jejuni, C. coli Enteropathogenic, enterotoxigenic and enteroinvasive Escherichia coli Enterohemorrhagic E. coli Legionella spp. Nontuberculous mycobacteria Pseudomonas aeruginosa Salmonella typhi Other Salmonellae Shigella spp. Vibrio cholerae Yersinia enterocolitica

High High Low Moderate High High High High High

Adenoviruses Enteroviruses Hepatitis A virus Hepatitis E virus Norwalk virus Noroviruses and sapoviruses Rotaviruses

High High High High High High High

Acanthamoeba spp. Cryptosporidium parvum Cyclospora cayetanensis Entamoeba histolytica Giardia intestinalis Naegleria fowleri Toxoplasma gondii


Dracunculus medinensis Schistosoma spp.

High High

Low High High

Persistence in Water Bacteria May multiply Moderate Moderate

Resistance to Chlorine

Relative Infective Dose

Low Low Low

Low Moderate Low

Low Low High Moderate Low Low Low Low Low

High Moderate Low Low Low Low Moderate Low Low

Moderate Moderate Moderate Moderate Moderate Moderate Moderate


Protozoa Long Long Long Moderate Moderate May multiply Long



Helminths Moderate Short

Moderate Moderate

High High

Moderate Multiply Multiply May multiply Moderate May multiply Short Short Long Viruses Long Long Long Long Long Long Long

Source: World Health Organization (WHO), Guidelines for Drinking-Water Quality, Vol. 1, Recommendations, 3rd edn., World Health Organization, Albany, NY, 2006.

16


aeruginosa, and species of Flavobacterium, Acetinobacter, Klebsiella, Serratia, and Aeromonas can cause illness in people with impaired immunity. The microbiological quality of water is monitored throughout different stages of its distribution by testing for the coliform group or fecal coliforms (E. coli), which are indicators of fecal contamination. Most of the pathogens listed in Table 1.3 are so infective that the ingestion of even one viable organism could lead to the contraption of the disease, so the presence of any fecal contamination is unacceptable in drinking water. Therefore, the acceptable level of coliforms and fecal coliforms is zero. The tests are based on the presence or absence of the organisms; however, the regulatory limits allow for 5% of monthly analyzed samples to test positive. The sampling frequency is predetermined depending on the number of consumers. The conventional methods for the analysis of microbes in water depend on culturing the organisms followed by application of biochemical tests for identification. In the past decade, rapid methods of detection of these organisms have been achieved by a number of newly developed methods. The surface plasmon resonance (SPR), amperometric, potentiometric, and acoustic wave sensors and their applications in biosensor systems are used for the detection of pathogens [51]. Biomolecular techniques based on the polymerase chain reaction to amplify genetic material have also been applied as rapid methods for detection. The PCR based methods have been successfully applied for virtually all types of pathogens listed in Table 1.1 and provide greater sensitivity, specificity, and accuracy with the drawbacks of being expensive and complicated [51,52]. Turbidity is a physical parameter monitored in relation to the microbiological quality of drinking water. The aim of monitoring turbidity is to reduce interference of particulate matter with disinfection by sheltering microorganisms, maintenance of chlorine residual, and problems that can arise in microbiological testing resulting from high bacterial populations. Suspended matter, even if biologically inert, can eventually settle out causing silting and anaerobic niches in waterways. Turbidity in water is also an indicator of poor treatment due to improper operations or inadequate facilities. Blooms of algae in water are also undesirable because their proliferation results in technical problems as well as depreciation of the aesthetic qualities of the water. On the other hand, there are species of algae (i.e., Cyanobacteria spp.) which release hepatotoxic compounds into the water they grow in. The analysis of the toxins and the treatment of waters containing them are not practically possible, so suppliers must constantly monitor reservoirs for the development of algae. Waters containing excess amounts of nitrates and phosphates, which are nutrients for algae, are more susceptible for sustaining algal growth. There are no general or standard methods employed in the disinfection of water. Water disinfection methods must be decided on after a thorough evaluation of the source water and the route of delivery. Water treatment is a multiple barrier system and the terminal disinfection step should be applied to water that is nearly completely free of pathogens, pollutants, and biodegradable products. The majority of pathogens (>99%) are successfully removed by the coagulation, flocculation, sedimentation, and filtration steps during treatment. The primary purpose of disinfection is to kill or inactivate the remaining pathogens. The second purpose is to provide a disinfectant residual in the finished water and prevent microbial regrowth in the distribution systems. Chlorine, chloramines, ozone, chlorine dioxide, and ultraviolet radiation are the common disinfectants used in water treatment plants. The use of electrolyzed water is also gaining popularity as a sanitizer in the food industry. The acidic and basic fractions obtained by the electrolysis of dilute NaCl solution are applied on food surfaces to reduce the microbial loads [53]. 1.5.1.2 Chemical Parameters Water resources may contain a wide range of harmful and toxic compounds. Maximum levels and recommended guide values for selected contaminants are shown in Table 1.4.


17

TABLE 1.4 Selected Primary Maximum Contaminant Levels in Potable Water Determined by the USEPA, the Equivalent Limits and Recommended Guideline Values Set by the EU Directive (98/83/EC), and the WHO Contaminant

USEPA

EU

Antimony Arsenic Asbestos Barium Beryllium Cadmium Chromium (total) Cyanide Fluoride Lead Mercury (inorganic) Nitrate Nitrite Selenium Thallium

Inorganic Chemicals 0.006 0.005 0.05 0.01 7 MFLb n.l.c 2 n.l. 0.005 n.l. 0.005 0.005 0.1 0.05 0.2 0.05 4 1.5 0.015 0.01 0.002 0.001 10 50 1 0.5 0.05 0.01 0.0005 n.l.

0.020 0.01 (P)a n.l. 0.7 n.l. 0.003 0.05 (P)a 0.07 1.5 0.01 0.006 50 0.2 (P)a 0.01 n.l.

Total coliform Turbidity

Microbial Factors 70% inulin) has been used as an ingredient without further processing [28]. 2.3.3.3 Food Uses Fructans, inulin, and FOS have food ingredient status in most countries and are recognized as GRAS ingredients in the United States. In general, fructans have a bland neutral taste with no off-flavor or aftertaste [27,30]. Fructans are used in a wide range of food products such as bakery products, breakfast cereals, drinks, and dairy products. Fructans are heat labile and will break down under low pH [30,31]. Fructans have humectant properties that reduce water activity and improve microbiological stability. When mixed with water, inulin can form a particle gel network, resulting in a white creamy structure with a short spreadable texture [32]. Emulsion of long-chain fructan in water has organoleptic properties similar to fat and has been used as a fat replacement in food systems. Inulin works in synergy with most gelling agents. FOS are very water soluble and have some sweetness (30%–35% of sucrose) [27]. FOS are used in beverages where they improve mouthfeel, enhance fruit flavor, and sustain flavor with less aftertaste when added to artificial sweeteners, aspartame or acesulfame K.

Glycobiology of Foods

31

2.3.3.4 Healthful Properties Fructans are a recognized form of dietary fiber. Digestive enzymes in the human small intestine are specific for α-glycosidic linkages. Thus the β-configuration of the anomeric carbon makes fructans resistant to hydrolysis in the human small intestine [14]. Kolida et al. [33] summarized several studies that document the prebiotic effect of fructans. Fructans are an efficient carbon source of beneficial Bifidobacteria in the colon, which enhances the growth of healthy gut microflora while suppressing the growth of pathogenic bacteria [25,32,33]. Fructans promote mineral absorption, particularly calcium [14], enhance immune functions and antitumor activity, and may modulate lipid and carbohydrate metabolism for good cardiovascular and diabetic health [30].

2.4 POLYSACCHARIDES 2.4.1

GENERAL

Polysaccharides are composed of more than 20 monosaccharide units. Polysaccharides often are classified as being starch or nonstarch. Starch polysaccharides represent a source of energy in human beings while nonstarch polysaccharides generally are nondigestible and are important in maintaining intestinal health.

2.4.2

STRUCTURE

Polysaccharides are a very diverse set of compounds. They can consist of one type of sugar (homoglycans) or several types of sugars (heteroglycans). They can be branched or nonbranched and can vary in type of linkage. The three dimensional structure or conformation of polysaccharide chains is determined by the monosaccharide units and position and type of glycosidic linkages. For example, polysaccharides with α-(1-4)-d-glucosyl linkages or β-(1-3)-d-glucosyl linkages form hollow helixes while those with β-(1-4)-d-glucosyl linkages form ribbonlike conformations. The ribbonlike and hollow helix conformations are the two basic chain conformations. Polysaccharides associated with cell walls generally have a ribbonlike conformation. The ribbon conformation allows the chains to align parallel to each other. This promotes efficient packing and strong hydrogen bonds, which in turn allows them to form fibrous water insoluble aggregates. All ribbonlike conformations have some degree of zigzag structure. Hollow helices tend to be more flexible than ribbonlike conformations. The flexibility is reduced by formation of an inclusion complex with another molecule (e.g., amylose–lipid complex) or by combining to form multiple helixes or nesting helical conformations.

2.4.3

WATER SOLUBILITY

Unsubstituted glycosyl units contain three hydroxyl groups and one ring oxygen all of which can hydrogen bond with other polymers or with water. Hydrogen bonding has a major influence on the water solubility of polysaccharides. During hydration, the hydrogen bonding changes from hydroxyl groups of adjacent glycan molecules to hydrogen bonding with water, the reverse is true during dehydration. Solubility of polysaccharides depends on intermolecular hydrogen bonding and whether steric hindrance (often in the form of branches) keeps the chains at a distance from each other so that water can penetrate and hydrate the polymers. Single, neutral monosaccharide units with one type of linkage and few or no branches are usually insoluble in water and are difficult to solubilize. They form orderly conformation within the chain and chain–chain interaction. Branched polysaccharides are more water soluble. Branches reduce chain–chain interaction, which allows easier hydration. Dried branched polysaccharides rehydrate more quickly than do dried nonbranched polysaccharides.

32


2.4.4

VISCOSITY

Most polysaccharides increase solution viscosity. Solution properties of a polysaccharide depend on its structure, molecular weight, and concentration. Linear molecules in solution sweep out a large space, which increases their probability of colliding with other molecules and forming a viscous solution. In contrast, highly branched molecules of the same molecular weight sweep less space and form less viscous solutions. Nonstarch polysaccharides generally have Newtonian flow properties at low concentrations and non-Newtonian flow behavior at moderate to high concentrations. Flow is based on the cohesiveness of the mixture of molecules with different shapes and sizes and how much force is required to move them.

2.4.5

GEL FORMATION

Gel formation requires a balance of chain homogeneity with chain irregularities. Thus, many polysaccharides do not form gels but form entanglement networks. A homoglycan polysaccharide will tend to associate greatly with other chains and precipitate out of solution. Chains that allow no chain–chain interaction cannot form a gel since they are unable to form junction zones necessary to build a three-dimensional network. Thus, chains that allow some chain–chain interaction are more likely to form a gel. The region of chain–chain interaction (junction zones) is often terminated by structural irregularities which prevent complete aggregation and precipitation of the polysaccharide. For this reason it is not uncommon for a mixture of polysaccharides to form a gel even though singly they are unable to. For example, locust bean gum (nongelling) and xanthan gum (weak gel) together form a strong gel. This is attributed to the interaction among different chain polymers and formation of mixed junction zones.

2.4.6

STARCH

2.4.6.1 Occurrence in Plants The primary function of starch is to serve as energy storage and as a carbon source for de novo biosynthesis of macromolecules. Starch can accumulate temporarily in the chloroplast of cells found in photosynthetic tissue. Most starch is found in the storage organs such as the endosperm of seeds or in roots and tubers. Starch is composed of two polysaccharides: amylose and amylopectin. Amylose and amylopectin are both polymolecular [contain α-(1-4) and α-(1-6)-d-glucosyl linkages] and polydisperse (vary in degree of polymerization). Amylopectin is a large, highly branched molecule with a degree of polymerization of 104 –107 with 4%–5% of the linkages involved with branch points (Figure 2.6) [34]. Amylopectin branches are categorized as A-chains, B-chains, and C-chains. H CH2OH H O

HO

H HO

H HO

H CH2OH H O OH

H

H

CH2OH H O H H

FIGURE 2.6

O CH2

H

OH

O HO

H

CH2OH H O

H O OH

H

H HO

H

H H

Amylopectin.

OH

H

H

CH2OH H O

O HO

O

H H

CH2OH H O

O H

OH

H

H

H H

HO

H O

HO

HO

OH

H

H

CH2

H O

HO

H H

OH H

CH2OH H O

O

O HO

H H

OH

O HO

H

H H

OH

H

Glycobiology of Foods

33 H

H CH2OH H

O

HO

CH2OH H

H O

O HO

H

FIGURE 2.7

H

H H

H

H O

O HO

OH

CH2OH H

OH H

CH2

H

O O

O HO

H H

OH H

HO

H

OH H

H

Amylose.

The C-chain is the original chain. B-chains are branch chains that have branches. A-chains are branch chains that are not branched; hence, they are the outermost chains. Amylose is smaller than amylopectin with a typical degree of polymerization of 103 –104 and is nearly nonbranched with only 0.3%–0.6% of the linkages involved with branch points (Figure 2.7) [34]. In general, amylose comprises 20%–30% of the starch, although high (≈65%) and low ( disaccharides. Fructose did not condense with amino acids in dilute solution although scientists have since confirmed that a definite interaction does take place. d-Fructose has also been reported to brown at a much faster rate than glucose during the initial stages of the browning reaction, but it then falls behind. This was confirmed using model systems containing glucoseglycine and fructose-glycine [189]. Sucrose, as a nonreducing sugar, will only participate when the glycosidic bond is hydrolyzed and the reducing monosaccharide constituents released. Hydrolysis of the glycosidic bond in sucrose is facilitated by a low pH, resulting in an increase in the Maillard reaction rate in protein–sucrose systems. Claeys et al. [190] studied the kinetics of acrylamide by heating a model system consisting of asparagine and glucose, fructose, or sucrose (pH 6) at temperatures between 140°C and 200°C. Acrylamide formation appeared to proceed faster and to be more temperature sensitive in the asparagine glucose than in the asparagine-fructose model system. Significantly less acrylamide was formed in the asparagine-sucrose model system as compared to the model systems with glucose or fructose. 11.3.1.2.3 Sugar:Amine Ratio The extent of browning seems to vary to the initial concentrations and ratio of the reactants. O’Brian and Morrisey [172] stated that, an excess of reducing sugar over amino compound promotes the rate of Maillard browning, since there are mechanistic differences in the destruction of sugar compared

Enzymatic and Nonenzymatic Browning Reactions and Control Mechanisms

363

to the amino acid. However, Warmbier et al. [191] found that, the browning rate increased to a maximum at glucose:lysine ratio of 1:3 (present in casein). Since, the initial step of formation of the Schiff base is dependent on the concentration of both, sugar and amino acid, the Schiff base formation increases with decreasing sugar:amino acid ratio. Labuza and Baisier [186] showed that the ratio of amino acid to reducing sugar can affect the rate of browning. The effect of increasing the amino acid concentration shows a greater increase in browning than that of increasing the sugar content on a molar basis and the increase for both is greater than the relative concentration increase. Renn and Sathe [192] investigated the effects of pH, temperature, and reactant molar ratios on the l-leucine and d-glucose Maillard browning reaction in an aqueous system. At 100°C and pH 9 and 10, samples with excess leucine had higher mean values for color than the mean color values for samples using glucose/leucine ratios of 1:1 or 2:1. At 122.5°C and pH 9 and 10, samples with glucose/leucine ratios of 1:2 had a significantly higher rate of glucose loss than samples containing glucose/leucine ratios of 2:1 and 1:1 at the corresponding pH. Kato et al. [193] observed that, at low concentrations of glycine, fructose browned faster than glucose, whereas, at high amino acid concentration, the reverse occurred. However, Martins and van Boekel [194] pointed out that the rate of browning and the rate constant of the step in the reaction network that results in color formation, are two different things. They showed that increasing the initial reactant concentrations should not influence the reaction rate constants since, for the reaction of sugar (S) with the amino acid (A), it follows that −

d[S] d[A] =− = k1[S][A] dt dt

The overall rate of loss of the S and the A is equal to the rate constant times their concentration. So, the overall rate depends on the concentration. 11.3.1.2.4 Effect of pH Both the initial pH of the product and the buffering capacity of the system, influence the rate and direction of the Maillard reaction. Borsook and Wasteneys [195] were the first to investigate systematically the influence of pH on the extent of interaction of glucose and free amino-nitrogen. They made quantitative observations on the interaction of glucose with glycine, and of glucose with various enzymatic digests of protein material. Determinations of pH and Van Slyke amino nitrogen were made with time; in each case the initial pH values were set by phosphate buffers and the solutions contained 1% glycine and 13.2% glucose. An appreciable loss of amino-nitrogen occurred in 48 h; the loss increased with increasing pH. It was shown that the optimum pH for the whole reaction is in the range pH 6–9 [4]. With regard to the influence of pH on the Maillard reaction, Labuza and Baisier [186] observed that the substrate loss increased with increasing pH, up to a pH of about 10, with little, if any, browning occurring below pH 6. The rate of the reaction is also dependent on the concentration of the acyclic sugar present. The amount of acyclic sugar increases with increasing pH and so increases the rate of reaction. The formation of the enediol anion is also considered a key reaction in both reversible and irreversible sugar reactions. Reversible sugar reactions are ionization, mutarotation, enolization, and isomerization, and these reactions imply that the sugar moiety remains intact. Irreversible sugar reactions imply that the sugar moiety is eventually degraded in organic acids. Since ionization is a rate-determining step, pH obviously has an influence on sugar reactions and hence on the Maillard reaction [194]. Furthermore, the main degradation pathways of the Amadori compound, namely enolization and retro-aldolization, were shown to be strongly dependent on the reaction pH [196]. The pH-dependence of the Maillard reaction for the amino acid reagent can, at least qualitatively, be described by the effect of protonation of the amino acid. The amount of unprotonated amino group, which is considered to be the reactive species, increases obviously with increasing pH.

364


The nonenzymatic browning reactions of fructose and fructose-lysine aqueous model systems were investigated at 100°C between pH 4.0 and pH 12.0 by measuring the loss of reactants and monitoring the pattern of UV-absorbance and brown color development [2]. At all the pH values tested, the loss of fructose was lower in the presence than in the absence of lysine. The promoting effect of pH was clear on the browning development and was in agreement with the earlier studies [4,197]. Lertittikul et al. [198] monitored the browning products by heating the solution containing 2% porcine plasma protein (PPP) and 2% glucose adjusted to various pHs (8, 9, 10, 11, and 12) at 100°C for different times (0, 2, 4, 6, and 8 h). Among all the MRPs tested, those derived from the PPPglucose system at pH 12 rendered the highest browning and intermediate products. However, an electrophoretic study revealed that cross-linked proteins with high molecular weight were formed in the PPP-glucose model system to a greater extent at pHs 8 and 9, than at pHs 10–12. The pH-dependence of acrylamide formation exhibited a maximum around pH 8; lower pH enhanced elimination and decelerated the formation of acrylamide in heated foods [199]. 11.3.1.2.5 Temperature Effect The most important influence on the kinetics of the Maillard reaction is temperature. Increasing temperature results in a rapidly increasing rate of browning. Not only the rate of browning but also the character of the reaction is determined by temperature. In model systems, the rate of browning increases 2–3 times for each 10° rise in temperature. In foods containing fructose, the increase may be 5–10 times for each 10° rise. Temperature also affects the composition of the MRPs formed. If the color intensity is measured, it may also be increased with increasing temperature because of the changing composition and increasing carbon content of the pigment. Benzing-Purdie et al. [200] worked with equimolecular amounts of d-xylose and glycine in aqueous solution at temperatures of 22°C, 68°C, and 100°C. They reported that an increase in temperature leads to an increase in aromatic character in both high and low molecular weight products. The structure of the melanoidins synthesized at room temperature differs considerably from those synthesized at higher temperatures in that they have different types of aliphatic carbons and fewer unsaturated carbons. As with other chemical reactions, the Arrhenius relationship provides a good description of the temperature dependence of the Maillard browning reaction: k = k0 exp( − Ea /RT ) where k is the reaction rate constant of nonenzymatic browning k0 a frequency constant (independent of temperature) Ea the activation energy R the universal gas constant T the absolute temperature Linear, exponential, and hyperbolic functions have also been employed to correlate browning but were found to be valid only over a limited range. Quantitative knowledge is very important in order to control the Maillard reaction in food processing operations. However, it should be noted that the various reaction steps have different temperature sensitivities. Therefore, it is a necessary first to specify what has to be measured for the determination of the activation energy of the Maillard reaction. In most cases, the reported activation energies will reflect more elementary reaction steps, and will be the resultant of one or more rate-controlling steps. The variation in activation energies reported in the literature is vast. Unfortunately, activation energies reported for the same reaction step can easily differ by a factor of 4.


365

This probably reflects the importance of the experimental conditions: mechanisms may change depending on these conditions, and this will be reflected in the activation energies [201]. Activation energies can be determined for browning reactions in various food systems as a function of water content, from which the temperature dependence of the browning reaction at different water contents can be elucidated. Miao and Roos [202] studied the effects of water contents on nonenzymatic browning (NEB) rates of amorphous, carbohydrate-based food model systems containing l-lysine and d-xylose as reactants at different temperatures (40°C, 50°C, 60°C, 70°C, 80°C, and 90°C) of spray drying. They modeled the data by using the Brunauer-Emmett-Teller and Guggenheim-Anderson-deBoer equations. The rate of browning increased with water content and temperature, but a lower (T − Tg) was needed for browning at decreasing water content. Water content seemed to affect the activation energy of NEB, and higher water contents decreased the temperature dependence of the NEB. At higher temperatures, the NEB became less water content dependent and enhanced the browning in spray-drying. The temperature dependence of nonenzymatic browning could also be modeled using the Williams-Landel-Ferry (WLF) equation, but the WLF constants were dependent on the water content. Bozkurt et al. [203] evaluated the reaction orders, rate constants, and activation energies for HMF accumulation and brown pigment formation (BPF) in boiled grape juice and its model systems at 55°C, 65°C, and 75°C over 10 days at pH 4.0. The calculated activation energies for HMF accumulation and brown pigment formation were in the range of 49.7–103 kJ/mol and 116–132 kJ/ mol, respectively. Gög˘üs¸ et al. [182] studied the effect of temperature in various model systems containing amino acids in single or in combination, in the presence of glucose and fructose. They found that the rate of browning increased 3.20 times from 55°C to 65°C and 3.50 times from 65°C to 75°C in a glutamine (0.015 mol/L), glucose (2.0 mol/L), and fructose (2.0 mol/L) model at pH 3.5 (Figure 11.13). Pedreschi et al. [204] investigated acrylamide formation and changes in color of fried potato strips in relation to frying temperature. Acrylamide formation decreased dramatically as the frying temperature decreased from 190°C to 150°C. Color showed high correlations with French fry acrylamide content. A paper summarized the characterization of parameters that influence the formation and degradation of acrylamide in heated foods [199]. It has been found that the higher temperature (200°C)

12 55°C 65°C 75°C

Absorbance at 420 nm

10 8 6 4 2 0 0

2

4

6

8

10

12

Time (days)

FIGURE 11.13 Effect of temperature on brown pigment formation in a “glucose + fructose + glutamine” model system (G, glucose; F, fructose; Gln, glutamine).

366


combined with prolonged heating led to reduced levels of acrylamide, due to the elimination and degradation processes. At certain concentrations, the presence of asparagine or monosaccharides (in particular fructose, glucose, and glyceraldehyde) was found to increase the net content of acrylamide. Addition of other free amino acids or a protein-rich food component strongly reduced the acrylamide content, probably by promoting competing reactions and/or covalently binding the formed acrylamide. 11.3.1.2.6 Water Activity Nonenzymatic browning reactions may occur as a result of heating, dehydrating, or concentrating food constituents. The role of bound and unbound water in browning reactions has been investigated by several authors. Almost all of the results showed that a maximum browning rate occurs at water activities between 0.4 and 0.6 depending on the type of the food substance. At lower water activities the reaction rate decreases as a result of the increasing diffusion resistance due to high viscosity [205], whereas at higher water activities the reaction rate again slows down, due to the dilution of the reactants [206]. Furthermore at higher water activities, water behaves as a reaction product and blocks the formation of reaction intermediates that are produced together with water. Labuza et al. [206] explained the properties based on the water that plays a role in the chemical reactions: (1) water as a solvent in which reactants are able to dissolve, be transported, and react; and (2) water as a reactant which participates in specific reactions. According to their investigations, even at low water activities sucrose may be hydrolyzed to form reducing sugars which have a potential for browning. They have found that the formation of browning products of dehydrated skim milk at 54°C is the maximum at 75% relative humidity, and the rate decreases with decreasing relative humidity. They proposed that water not only accelerates the reaction, but also shortens the induction period. The acceleration of the rate may be due to the increased availability and mobility of the reactants. The effect of induction time may indicate that the formation of pigment has a different pathway at the lower humidity, which requires more time for color development, or that sufficient intermediates must be built up before they can dissolve and react to form the pigment [206]. They have also examined browning products formation in pea-soup at a wide range of water activities from 0.0 to 0.9 again the browning rate was the maximum at a water activity of 0.75. At higher water activities the rate of reaction decreased sharply. The major reason is that at this point the maximum amount of reactants can be dissolved in solution; the problem is no longer diffusion, but rather that as water content increases the reactants are diluted. Since by the law of mass action, the rate of a reaction is proportional to concentration, a decrease in concentration by the dilution with water will decrease the rate. Warmbier et al. [191] have studied the effect of water activity on BPF by using a humectant, glycerol. They prepared a model system containing K-sorbate, glucose, casein, aprezon-B oil, microcrystalline cellulose, and variable amounts of water. They measured the browning rate at 420 nm and found that there is a linear relationship between time and BPF. The maximum rate of nonenzymatic browning occurred at water activities 0.45–0.55. They also found that when the temperature increases the browning maximum shifts to higher water activities. They proposed that liquid humectant (glycerol) can cause the water activity for the maximum browning to be shifted downward. Another important study on brown pigment formation and water activity was carried out by Eichner and Karel [205] on model systems containing variable amounts of microcrystalline cellulose MCC, methylcellulose, glycerol, and gum arabic together with the reducing sugar (glucose) and amino acid (glycine). They found that addition of the MCC to the original system containing sugar, amino acid, glycerol, and water had no effect on the browning rate, whereas the system with gum arabic showed the lowest rate. Gum arabic also shifted the water activity for maximum browning upward. At high water activities (aw > 0.75) all of the hydrocolloids showed similar behavior. Finally they concluded that browning rate in a sugar-amino system is not simply related to the water


367

activity. Optimum browning conditions are determined by either the amount of water, or the state of water binding in a distinct system. Eichner [207] have studied the effect of water activity in the different steps of the Maillard mechanism by determining the reaction intermediates in a model system containing glucose-lysine (1:1 mol) and MCC (14 g/g glucose) at 40°C, at water activities between 0.23 and 0.82. He found that (1) the decrease in the free amino group of lysine was minimum at aw 0.23, and maximum around water activities 0.62–0.75; (2) not only the rate of formation of reducing browning intermediates, but also their rate of decomposition appeared to increase at higher water activities and the nature of the reducing intermediates changed due to a change in water activity; and (3) at higher water activities, browning was proceeding directly via 1,2 eneaminol intermediate of the Amadori rearrangement, without using the Amadori rearrangement products (ARPs). The effect of water activity in a range between 0.11 and 0.80 was observed by measuring both BPF and HMF accumulation in concentrated orange juice with and without xanthan gum and microcrystalline cellulose [208]. They reported that the samples containing 1.5% xanthan gum and 0.5% microcrystalline cellulose showed the lowest BPF and HMF accumulation, whereas the browning rate and HMF accumulation in pure orange juice concentrate were the highest at all temperatures studied (45°C, 60°C, and 75°C). Figure 11.14 shows the effect of water activity on both HMF accumulation and browning of pure orange juice concentrate after 48 days of storage at 45°C. It is seen that both HMF accumulation and browning show a maximum at aw 0.50. However, HMF accumulation has a wider range of maximum compared to the sharp maximum of browning. Ameur et al. [209] determined the critic aw value where HMF began to form, to describe better the relationship between the water activity and the formation of HMF. This HMF level was defined as the analytical limit of quantification of 3.60 mg/kg. They found that the water activity allowing HMF formation was significantly higher at 300°C (critical aw of 0.7) than at 250°C or 200°C (mean critical aw of 0.51). Mundt and Wedzicha [210] also explored the effect of aw on the rate of browning in biscuit doughs at 105°C–135°C. They found that, contrary to common expectation, the value of aw in the range 0.04–0.4 did not affect the measured rate of browning. They concluded that this result has significant implications to the understanding of water relations at high temperatures, where the thermodynamic parameters governing water sorption are generally inaccessible.

400

70 Absorbance at 420 nm mg HMF/100 g

350

HMF (mg/100 g)

300 50 250 40 200 30

150

20


60

100 50

10 0.0

0.2

0.4

0.6

0.8

1.0

Water activity

FIGURE 11.14 Influence of water activity on HMF accumulation and brown pigment formation in orange juice concentrate at 45°C for 48 days.

368


11.3.1.3 Control Mechanisms of the Maillard Reaction The Maillard reaction is desirable in terms of the formation of color and flavor. However, in many other instances, such as fruits, vegetables, frozen, and dehydrated foods, browning is undesirable as it results in off-flavors and colors with the loss of nutritive value. It can also cause the formation of some toxic and mutagenic compounds in some cases. Therefore, it is important to know how to control or minimize the Maillard reaction in food processing. A variety of methods have been proposed for controlling the Maillard reaction. The Maillard reaction can be controlled or inhibited by controlling temperature, time, water activity, and pH; reduction of reducing sugar and/or amino nitrogen content; packaging with gas; application of high hydrostatic pressure, and the use of chemical inhibitors such as sulfites, flavonoids, and cations. 11.3.1.3.1 Temperature The rate of browning increases with increasing temperature or time. Since these reactions have been shown to have a high temperature coefficient, lowering of the temperature during the processing or the storage of food products can help to minimize these reactions. 11.3.1.3.2 Water Activity The Maillard reaction being moisture dependent for optimum activity, it can be inhibited by reducing the moisture content through dehydrating procedures. Sherwin and Labuza [211] showed the role of moisture in the Maillard browning reaction rate in intermediate moisture foods. They found that the addition of humectants to a model formulation caused significant plasticization and a lowering of the Tg curve. However, with the addition of the sorbitol, a solid-state humectant, there was no change in reaction rate at equal moisture content, in comparison with the control. For the glycerol formulation, a liquid phase humectant, there were high rates of browning, between aw = 0.11 to 0.75, that were greater than for the control formulation. They concluded that a solvency-based molecular mechanism describes the effect of increased moisture on reaction rate in a semi-moist food. 11.3.1.3.3 pH The pH of the Maillard browning reaction is an important parameter for both the reaction rate and the characteristics of the products. Since the Maillard reaction is generally favored at the more alkaline conditions, lowering of the pH might provide a good method of control. 11.3.1.3.4 Biochemical Agents Removal or conversion of one of the reactants of the Maillard reaction controls the browning. For instance, glucose or other fermentable sugars in liquid egg whites, egg yolks, or whole eggs can be removed by yeast or bacterial fermentations. The use of commercial glucose oxidase-catalase preparations is a widely used alternate commercial method for removing glucose. Gluconic acid produced from glucose is acceptable in small concentrations. This enzyme has been used to remove glucose from egg prior to spray drying [7]. Another study has been performed to control the Maillard browning in fried potato chips by yeast fermentation [212]. They studied the effect of different combinations of yeast concentration and fermentation time. They observed that browning of chips decreased 60% and the yield of acceptable chips increased considerably after the yeast treatment at the optimum yeast concentrations. 11.3.1.3.5 Modified Atmosphere Packaging Modified atmosphere packaging is useful in excluding oxygen by using an inert gas. This reduces the possibility of lipid oxidation, which in turn could give rise to reducing substances capable of interacting with amino acids. While this reaction does not appear to influence the initial carbonylamino reaction, exclusion of oxygen is thought to effect other reactions involved in the browning process [7]. Maillard reactions are the main causes of brown color formation in glucose syrup.


369

Effects of modified atmosphere packaging (MAP) on browning in glucose syrups stored at 25°C and 45°C were studied [213]. They examined different atmospheres such as air, 100% N2, 90% N2/10% O2, 25% CO2/75% N2, 75% CO2/25% N2, and vacuum. The oxygen content and pressure affected the Maillard reaction. They found that in the absence of oxygen, the browning rate in the glucose syrup samples was low while it increased with the increase in oxygen content. 11.3.1.3.6 High Pressure Another parameter controlling the Maillard reaction is high (hydrostatic) pressure. Moreno et al. [214] studied glucose-lysine model systems over a range of pH values (5–10) in unbuffered and buffered media at 60°C either under atmospheric pressure or at 400 MPa. They reported that in the buffered media, at pH values less than or equal to 8.0, the pressure slowed the Maillard reaction from the initial stages. These effects were attributed to the pH drop caused by the pressure-induced dissociation of the acid groups. However, they also noted that, in the unbuffered and buffered media at an initial pH = 10.2, the high pressure accelerated the formation and subsequent degradation of ARP, leading to increased levels of intermediate and advanced reaction products. Komthong et al. [215] also studied the effect of high hydrostatic pressure (100 MPa) combined with pH (6.0, 7.0, and 8.0) and temperatures (80°C and 90°C) on the Maillard reaction of the sugar (glucose or fructose)amino acid (leucine, lysine, or glutamate) solution models. They found that both the formation of browning products and HMF content decreased by the high pressure treatment. 11.3.1.3.7 Chemical Inhibitors In view of the wide occurrence of the Maillard reaction products during the production and storage of various different food products, it would be of great interest to limit this reaction in the undesirable cases. A variety of chemical inhibitors have been used for that purpose. Sulfites are the most widely used inhibitors. However, the restrictions to the use of sulfite agents in foods promoted the scientists to develop alternatives to sulfites. Therefore; calcium salts, thiols, aspartic and glutamic acids, phenolic acids, and various flavonoids have been studied as alternatives to sulfites. On the other hand, scientists have made efforts for finding the chemical way to reduce the amount of acrylamide in the Maillard reaction during the last few years. It has been reported that cations effectively prevented the formation of the Schiff base, which is the key intermediate leading to acrylamide, and mainly changed the reaction path toward the dehydration of glucose leading to HMF and furfural [216]. It has also been found that aminoguanidine could inhibit the Maillard reaction both in vitro and in vivo. The action of aminoguanidine is probably due to the trapping of intermediates of the advanced Maillard reaction, such as 3-DG, leading to the inhibition of further progress of the Maillard reaction [217]. Sulfites/Sulfur dioxides are commonly used as food additives. They are known as food preservatives but also have an important role as inhibitors of enzymatic and nonenzymatic browning. However, they are subject to regulatory restrictions because of their adverse effects on health. In the context of a food additive, “sulfur dioxide” refers to a mixture of oxospecies of sulfur in an oxidation state of (+4), i.e., SO2, HSO3− , SO32 − , S2O52 −. Sulfite and hydrogen sulfite ions are formed by the ionization of the species H2SO3, which is more correctly written as SO2⋅H2O. In practice it is very difficult to provide a quantitative description of these ionic and nonionic forms in foods. To avoid ambiguity the term S(IV) is used to describe the mixture of sulfur species when it is not necessary or possible to identify the detailed composition. Sulfur dioxide/Sulfites can be used to control nonenzymatic browning because of their ability to react with the carbonyl intermediates. A variety of carbonyl intermediates can be formed during the nonenzymatic browning process, including simple carbonyls, dicarbonyls, and a, b-unsaturated carbonyls. Sulfites can react with all of these intermediates and thus block the formation of the brown pigments. Sulfites inhibit the Maillard reaction by binding with the intermediates formed during the early stage of the reaction to form sulfonates. The important sulfonates are formed as a result of the replacement of the hydroxyl group at position 4 of the

370


3-deoxyhexosulose intermediate. In the Maillard reaction of glucose, 3-deoxyhexosulose (DH) is converted to 3,4-dideoxy-4 -sulfo-d-erythrohexosulose (DSH) [218]. The formation of DSH involves the conversion of DH to 3,4-dideoxyhexosulose-3-ene (DDH) which is probably also the most reactive known intermediate. Wedzicha and Vakalis [219] developed a kinetic model for the sulfite inhibition of the browning in the homogeneous aqueous “glucose + glycine + S(IV)” system. It was found that the fi rst determining step of this conversion, in fact, catalyzed by S(IV) in reactions leading to monofructoseglycine (MFG) or in the decomposition of MFG to DH. The formation of MFG is subject to general acid- or base-catalysis. It has been shown that sulfite and disulfite ions are capable of being effective acid-base catalysts. Flavonoids are known for their ability to control the Maillard browning. Colahan-Sederstrom and Peterson [220] studied the effect of Epicatechin (EC) to inhibit the thermal development of aromatic MRPs formed during ultrahigh-temperature (UHT) processing of bovine milk. They reported that the addition of EC to raw fluid milk prior to UHT processing reduced the overall thermal formation of key aroma-active compounds in comparison to the traditional UHT milk sample. Epicatechin (EC) and epigallocatechin gallate (EGCG) have also been used in another study to investigate their effect on Maillard browning in the UHT milk and glucose/glycine model system [221]. They found that, EC and EGCG reduced Maillard fluorescence at the 0.1 mmol/L level, while fluorescence was negligible with added flavonoids at 1.0 mmol/L in the model glucose/glycine system. When these flavonoids were added to milk, they reduced the production of the Maillard associated fluorescence with UHT processing. They also reported that EC and EGCG reduced the total color difference during thermal processing. The prevention of acrylamide formation has been widely studied following the discovery of the formation of acrylamide during the Maillard reaction [163,222,223]. Acrylamide has been classified as “probably carcinogenic to humans” (class 2A) by the International Agency for Research on Cancer [224]. Becalski et al. [163] reported a decreased acrylamide formation when adding rosemary herb to the oil used for frying potato slices. Relatively lower amounts of acrylamide after the addition of a flavonoid spice mix have also been reported by Fernandez et al. [223]. Lindsay and Jang [222] found the suppression of acrylamide formation by polyvalent cations and polyanionic compounds in fried potato products. Effect of NaCl in reducing acrylamide formation also has been evaluated in potato chips during frying [225]. The soaking of potato slices in NaCl solution before frying dramatically reduced acrylamide formation in potato chips in 90% of the chips in comparison to the control chips. Gokmen and Senyuva [226] recently reported that dipping potatoes into calcium chloride solution inhibited the formation of acrylamide by up to 95% during frying. They found that the cations prevented the formation of the Schiff base of asparagines and so, the formation of acrylamide. Kwak and Lim [227] investigated the effects of antibrowning agents (cysteine, glutathione, sodium sulfite, pentasodium tripolyphosphate, citric acid, and oxalic acid) and phenolic acids (ferulic, hydroxybenzoic, syringic, and vanillic acids) on the inhibition of browning in a glucose-lysine model system. They found that citric acid was the most efficient antibrowning agent during storage in air at 30°C and inhibited browning to 36% after 4 weeks. They also reported that its antibrowning capacity was increased by 8%–15% in the presence of any of the phenolic acids.

11.3.2

CARAMELIZATION

The degradation of sugars in the absence of amino acids and proteins by heating them over their melting point and thereby causing color and flavor changes is called caramelization. If this reaction is not carefully controlled it could lead to the production of unpleasant, burnt, and bitter products. If the reactions are carried out under controlled conditions, pleasant qualities of caramel are obtained. During caramelization several flavor components as well as polymeric caramels are produced. Caramels are a complex mixture of various high molecular weight components. They can


371

be classified into three groups: caramelans (C24H36O18), caramelens (C36H50O25), and caramelins (C125H188O80). These polymers are often used as colors in commercial food products, from soft drinks to soy sauce, confectionary, and ice-cream. They are labeled as E150. Commercial caramels are produced directly by heating sugar, or by heating sugar in the presence of co-factors, such as ammonia or sulfite. This results in caramels with different colors or charged caramels. These aspects are very important for the use of different caramels in foods. Caramels used to color soft drinks should be negatively charged to prevent reactions with phosphates that cause precipitation and loss of color. On the other hand, caramels used for bakery goods should be positively charged. Caramelization starts at relatively high temperatures as compared to the other browning reactions, and depends on the type of sugar. In foods often several different carbohydrates and other components are present; all these may influence the caramelization temperature as well as the different steps and reactions, and thus the final flavors and colors that are produced. Diacetyl is an important flavor compound, produced during the first stages of caramelization. Besides diacetyl, hundreds of other flavor compounds are produced, for instance furans like HMF and hydroxyacetylfuran (HAF), furanones such as hydroxydimethylfuranone (HDF), dihydroxydimethylfuranone (DDF), and maltol from disaccharides, and hydroxymaltol from monosaccharides. It has been found that the pyrolysis of sucrose, glucose, and starch all produced caramels of similar composition [228]. However, it has been established that the fragmentation of sugars occurs to a significant extent at pH values below neutrality [229] and increases considerably at high pH values and temperatures, yielding colored N-free polymers [230]. The degradation of sugars has been proposed in two ways, as acidic and alkaline. In acidic degradation, 1,2-enediol forms from the aldose or ketose after a series of dehydration reactions. If the initial sugar is a hexose, 1,2-enediol is converted to HMF. If it is a pentose, 1,2enediol is converted to 2-furaldehyde. 3-Deoxyaldose-2-ene, 3-deoxyosulose, and osulos-3-ene are intermediates in the acidic degradation of fructose. The last series of reactions include both fragmentation reactions (flavor production) and polymerization reactions (color production). In alkaline degradation, the initial reaction is the transformation via the 1,2- and 2,3-enediol. Under strong alkaline conditions, continuous enolization progresses along the carbon chain, resulting in a complex mixture of cleavage products such as saccharinic acid, lactic acid, and 2,4-dihydroxybutyric acid. The development of color is very complex and involves a series of polymerization reactions. Ajandouz et al. [2] studied the caramelization of fructose in both acidic and alkaline conditions. They found that at initial pH values ranging from 4.0 to 7.0, a progressive accumulation of the intermediate degradation products occurred as a function of time and no lag time was observed, whereas a high level of fructose degradation occurring during the initial stages of the heating period was reported at pH 8.0 and pH 9.0. They also found that the rate of the caramelization reaction of glucose increased exponentially from pH 7.0 up to pH 12.0, while a linear increase in the rate of the caramelization reaction of fructose occurred in the same pH range. The contribution of the caramelization reaction to the overall browning in model systems of glucose and fructose in the presence and absence of amino acids have been investigated by Bozkurt [231] (Figure 11.15). He found that the contribution of the caramelization of glucose and fructose to the overall browning of glucose, fructose, and glutamine changed between 25% and 50% according to the studied temperature (55°C–75°C). It was reported that the increase in temperature increased the contribution of caramelization. Caramelization of sucrose requires a temperature of about 200°C. Reactions involved in the caramelization of sucrose include mutarotation, enolization and isomerization, dehydration and fragmentation, anhydride formation, and polymerization. The extent to which the reaction occurs depends upon pH, temperature, and heating time. Sucrose, held at 160°C as a melt, will hydrolyze to glucose and fructose anhydride. The production of water and organic acids such as acetic, formic, and pyruvic during sucrose caramelization will enhance the hydrolysis. Hydrolytic products, glucose and fructose, are reactants in the formation of caramel and volatile flavor compounds

372

Advances in Food Biochemistry 3.0 G+F G + F + Gln


2.5 2.0 1.5 1.0 0.5 0.0 0

2

4

6 Time (days)

8

10

12

FIGURE 11.15 Contribution of caramelization of glucose and fructose on brown pigment formation at 65°C in a “glucose + fructose + glutamine” model system (G, glucose; F, fructose; Gln, glutamine).

[232]. Heating sucrose to 300°C in a stream of nitrogen produces volatile furan compounds; namely 2-methyl furan, furan, 2-hydroxyacetyl furan, and other volatile reaction products (methanol, acetone, acrolein, propanol, and acetaldehyde) [230]. A major compound (2-furfural) and volatile thermal degradation products (2-furyl methyl ketone, 2-furyl propyl ketone, methyl-benzo(b)furan, and 5-methyl-3-hydro-furan-2-one) were identified by heating sucrose in an open evaporation dish at 180°C for 90 min [233].

11.3.3 ASCORBIC ACID OXIDATION Chemically, l-ascorbic acid belongs to the family of carbohydrates, functionally it is classified as an organic acid and a reducing reagent, and physiologically the compound is attributed to the class of essential vitamins [234]. Ascorbic acid is an antioxidant but it contributes to browning of foods because it is easily oxidized and decomposed under common storage and processing conditions. For this reason, ascorbic acid is responsible for the browning of most of the fruit juices and concentrates. It has a special importance in the citrus industry. The factors affecting ascorbic acid degradation are pH, oxygen, ascorbic acid concentration, temperature, light, metals, citric acid, and so on. l-Ascorbic acid in its reduced form is of utmost importance in nutrition and food processing. However, once it has degraded, it results in the production of reactive carbonyl compounds that act as intermediates in nonenzymatic browning in foods. These reactive carbonyl compounds undergo further reactions leading to the formation of brown pigments. Studies showed that l-ascorbic acid decomposition resulted in furfural and HMF formation [235]. The reaction of ascorbic acid in fruit juices and concentrates is very much dependent on pH, as the browning process is inversely proportional to pH over a range of 2.0–3.5. Juices with a higher pH are much less susceptible to browning, for example, orange juice at a pH of 3.4. Below pH 4.0, browning is due primarily to the decomposition of ascorbic acid to furfural [235]. Some investigators reported that during the degradation of l-ascorbic acid, a decrease in pH due to the formation of carbonyl compounds was noted. The measurement of sample pH after storage showed a constant pH of 3.65 [181]. Monsalve et al. [236] studied the degradation of dehydroascorbic acid and chlorogenic acid, at pH 6, and at variable water activities and temperatures in model systems containing cellulose, dehydroascorbic acid and/or chlorogenic acid. They found that at 45°C nonenzymatic browning of dehydroascorbic acid-cellulose model system increased linearly through a certain time and then it

Enzymatic and Nonenzymatic Browning Reactions and Control Mechanisms CH3

CHO

CHO

C

O

C

O

C HO

CH3

C

O

O

CH

CH2

CH2OH

CH2OH

L-Threosone

Diacetyl

373

3-Deoxy-L-Threosone

CH3 C

O

CHO Methyl glyoxal

FIGURE 11.16

CHO CHO Glyoxal

l-Ascorbic acid degradation products (a-dicarbonyl compounds).

remains nearly constant. The data showed that there is an inverse relationship between the brown product formation and water activity, and between the temperature and ascorbic acid loss. It is accepted that there are two possible pathways, namely oxidative and nonoxidative, for the nonenzymatic browning reaction of l-ascorbic acid. In the course of the nonenzymatic browning reaction of l-ascorbic acid, a wide range of degradation products have been reported in the literature [234,235,237]. In the presence of oxygen, ascorbic acid is degraded primarily to dehydroascorbic acid via a monoanion. The lactone of dehydroascorbic acid is hydrolyzed to form 2,3-diketogulonic acid, which does not show vitamin C activity. Decarboxylation of 2,3-diketogulonic acid produces xylosone that is further degraded to reductones and furan compounds. Dehydroascorbic acid cannot be formed in nonoxidative conditions, where ascorbic acid can undergo hydrolysis. Furfural can be formed from ascorbic acid in both oxidative and nonoxidative conditions. Shinoda et al. [238] have proposed pathways for the formation of the furan precursors furfural and 2-furoic acid. They found that the formation of furfural from ascorbic acid in model orange juice was repressed by the presence of ethanol and mannitol acting as free radical scavengers. Schulz et al. [234] used dehydro-Shinoda-ascorbic acid, which is the first characteristic intermediate of l-ascorbic acid degradation via the oxidative route, as a starting material of the reaction to distinguish it from the nonoxidative pathway. They found that dehydro-l-ascorbic acid yielded five a-dicarbonyl compounds, namely glyoxal, methylglyoxal, diacetyl, l-threosone, and 3-deoxyl-threosone (Figure 11.16). They concluded that these a-dicarbonyl compounds are formed from l-ascorbic acid on the oxidative pathway. But, they pointed out that these products can also be produced via the nonoxidative route. They also found that 3-deoxy-l-pentosone is exclusively formed via the nonoxidative route.

REFERENCES 1. Maillard, L.C. Action of amino acids on sugars. Formation of melanoidins in a methodical way, Compt. Rend., 154, 66, 1912. 2. Ajandouz, E.H. et al. Effect of pH on caramelization and Maillard reaction kinetics in fructose–lysine model systems, J. Food Sci., 66, 926, 2001. 3. Gazzani, G. et al. Mutagenic activity of the Maillard reaction products of ribose with different amino acids, J. Food Sci., 52, 757, 1987. 4. Ashoor, S.H. and Zent, J.B. Maillard browning of common amino acids and sugars, J. Food Sci., 49, 206, 1984. 5. Martins, S.I.F.S., Jongen, W.M.F., and van Boekel M.A.J.S. A review of Maillard reaction in food and implications to kinetic modeling, Trends Food Sci. Technol., 11, 364, 2001.

374


6. Davidek, J., Velsek, J., and Pokorny, J. Development in Food Science: Chemical Changes during Food Processing, vol. 21, Elsevier Science Publishers, Amsterdam, The Netherlands, 1990, p. 117. 7. Eskin, N.A.M. Biochemistry of Foods, 2nd ed., Academic Press, New York, 1990, Chap. 5, p. 9. 8. Macrane, R., Robinson, R.K., and Saadler, M.J. Encyclopedia of Food Science, Food Technology and Nutrition, vol. 1, Academic Press Limited, London, U.K., 1993, p. 146. 9. Ames, J.M. Control of the Maillard reaction in food systems, Trends Food Sci. Technol., 1, 150, 1990. 10. Wijewickreme, A.N., Krejpcio, Z., and Kitts, D.D. Hydroxyl scavenging activity of glucose, fructose, and ribose–lysine model Maillard products, J. Food Sci., 64, 457, 1999. 11. Kroh, L.W. Caramelization in food and beverages, Food Chem., 51, 373, 1994. 12. del Buera, M.P. et al. Nonenzymatic browning in liquid model systems of high water activity: Kinetics of color changes due to caramelization of various single sugars, J. Food Sci., 52, 1059, 1987. 13. Shallenberger, R.S. and Birch, G.C. Sugar Chemistry, AVI Publishing Co., Inc., Westport, CT, 1975, p. 169. 14. Lee, C.Y. Browning reaction, enzymatic, in Wiley Encyclopedia of Food Science and Technology, Francis, F.J., Ed., John Wiley & Sons, New York, 1999, p. 208. 15. Mayer, A.M. and Harel, E. Polyphenoloxidases and their significance in fruits and vegetables, in Food Enzymology, Fox, P.F., Ed., Elsevier Applied Science, London, U.K., 1991, p. 373. 16. Whitaker, J.R. Enzymes, in Food Chemistry, Fennema, O.R., Ed., Marcel Dekker, New York, 1996, p. 431. 17. Rouet-Mayer, M.A., Ralambosa, J., and Philippon, J. Roles of o-quinones and their polymers in the enzymic browning of apples, Phytochemistry, 29, 435, 1990. 18. Whitaker, J.R. and Lee, C. Y. Recent advances in chemistry of enzymatic browning, in Enzymatic Browning and Its Prevention, Lee, C.Y. and Whitaker, J.R., Eds., American Chemical Society, Washington, DC, 1995, p. 3. 19. Richard-Forget, F.C. et al. Oxidation of chlorogenic acid, catechins, and 4-methylcatechol in model solutions by apple polyphenoloxidase, J. Agric. Food Chem., 40, 2114, 1992. 20. Weemaes, C.A. et al. Activity, electrophoretic characteristics and heat inactivation of polyphenoloxidases from apples, avocados, grapes, pears and plums, Lebensm.-Wiss-u-Technol., 31, 44, 1998. 21. Özog˘lu, H. and Bayindirli, A. Inhibition of enzymic browning in cloudy apple juice with selected antibrowning agents, Food Control, 13, 213, 2002. 22. Selles-Marchart, S., Casado-Vela, J., and Bru-Martinez, R. Isolation of a latent polyphenoloxidase from loquat fruit (Eriobotrya japonica Lindl.): Kinetic characterization and comparison with the active form, Arch. Biochem. Biophys., 446, 175, 2006. 23. Nunez-Delicado, E. et al. Characterization of polyphenoloxidase from Napoleon grape, Food Chem., 100, 108, 2007. 24. Wong, M.K., Dimick, P.S., and Hammerstedt, R.H. Extraction and high performance liquid chromatographic enrichment of polyphenoloxidase from theobroma cacao seeds, J. Food Sci., 55, 1108, 1990. 25. Erat, M., Sakiroglu, H., and Kufrevioglu, O.I. Purification and characterization of polyphenol oxidase from Ferula sp., Food Chem., 95, 503, 2006. 26. Cheynier, V., Osse, C., and Rigaud, J. Oxidation of grape juice phenolic compounds in model solutions, J. Food Sci., 53, 1729, 1988. 27. Richard-Forget, F.C. and Gauillard, F.A. Oxidation of chlorogenic acid, catechins, and 4-methylcatechol in model solutions by combinations of pear (Pyrus communis Cv. Williams) polyphenol oxidase and peroxidase: A possible involvement of peroxidase in enzymatic browning, J. Agric. Food Chem., 45, 2472, 1997. 28. Vamos-Vigyazo, L. Polyphenol oxidase and peroxidase in fruits and vegetables, Crit. Rev. Food Sci., 15, 49, 1981. 29. Degl’Innocenti, E. et al. Biochemical study of leaf browning in minimally processed leaves of lettuce (Lactuca sativa L. var. acephala), J. Agric. Food Chem., 53, 9980, 2005. 30. Degl’Innocenti, E. et al. Physiological basis of sensitivity to enzymatic browning in “lettuce”, “escarole” and “rocket salad” when stored as fresh-cut products, Food Chem., 104, 209, 2007. 31. Nicolas, J. et al. Polyphenols and enzymatic browning, in Polyphenolic Phenomena, INRA Ed., Paris, 1993, p. 165. 32. Robinson, D.S. Peroxidase and their significance in fruits and vegetables, in Food Enzymology, Fox, P.F., Ed., Elsevier, London, U.K., 1991, p. 399. 33. Amiot, J.M. et al. Phenolic compounds and oxidative mechanism in fruit and vegetables, in Phytochemistry of Fruit and Vegetables, Tomas-Barberan, F.A. and Robins, R.J., Eds., Claredon Press, Oxford, U.K., 1997, p. 51.


375

34. Tomas-Barberan, F.A. and Espin, J.C. Phenolic compounds and related enzymes as determinants of quality in fruits and vegetables, J. Sci. Food Agric., 81, 853, 2001. 35. Jaenicke, E. and Decker, H. Tyrosinases from crustaceans form hexamers, Biochem. J., 371, 515, 2003. 36. Zawistowski, J., Biliaderis, C.G., and Eskin, N.A.M. Polyphenoloxidase, in Oxidative Enzymes in Food, Robinson, D.S. and Eskin, N.A.M., Eds., Elsevier Science, New York, 1991, p. 217. 37. Marshall, M.R., Kim, J., and Wei, C.-I. Enzymatic Browning in Fruits, Vegetables and Seafoods, FAO, Rome, Italy, 2000, http://www.fao.org 38. Lerch, K. Tyrosinase: Molecular and active site structure, in Enzymatic Browning and Its Prevention, Lee, C.Y. and Whitaker, J.R., Eds., American Chemical Society, Washington, DC, 1995, p. 64. 39. Pourcel, L. et al. Flavonoid oxidation in plants: From biochemical properties to physiological functions, Trends Plant Sci., 12, 29, 2007. 40. Mayer, A.M. Polyphenol oxidases in plants and fungi: Going places? A review, Phytochemistry, 67, 2318, 2006. 41. Lerch, K. Neurospora tyrosinase: Structural, spectroscopic and catalytic activity, Mol. Cell. Biochem., 52, 125, 1983. 42. Huber, M., Hintermann, G., and Lerch, K. Primary structure of tyrosinase from Streptomyces glaucescens, Biochemistry, 24, 6038, 1985. 43. Yoshida, H. Chemistry of lacquer (urushi), J. Chem. Soc., 43, 472, 1883. 44. Bertrand, G. Sur la laccase et sur le pouvoir oxydant de cette diastase, CR Acad. Sci. (Paris), 120, 266, 1985. 45. Walker, J.R.L. Enzymatic browning in fruits: Its biochemistry and control, in Enzymatic Browning and Its Prevention, Lee, C.Y. and Whitaker, J.R., Eds., American Chemical Society, Washington, DC, 1995. 46. Mayer, A.M. and Staples, R.C. Laccase: New functions for an old enzyme, Phytochemistry, 60, 551, 2002. 47. Huttermann, A., Mai, C., and Kharazipour, A. Modification of lignin for the production of new compounded materials, Appl. Microbiol. Biotechnol., 55, 387, 2001. 48. Bligny, R. and Douce, R. Excretion of laccase by sycamore (Acer pseudoplatanus L.) cells. Purification and properties of the enzyme, Biochem. J., 209, 489, 1983. 49. Tezuka, K. et al. Occurrence of heterogeneity on N-linked oligosaccharides attached to sycamore (Acer pseudoplatanus L.) laccase of excretion, Biochem. Mol. Biol. Int., 29, 395, 1993. 50. Sato, Y. et al. Molecular cloning and expression of eight cDNAs in loblolly pine (Pinus taeda), J. Plant Res., 114, 147, 2001. 51. Ranocha, P. et al. Biochemical characterization, molecular cloning and expression of laccases a divergent gene family in poplar, Eur. J. Biochem., 259, 485, 1999. 52. Dean, J.F.D. and Eriksson, K.-E.L. Laccase and the deposition of lignin in vascular plants, Holzforschung, 48(Suppl.), 21, 1994. 53. Vaughn, K.C. and Duke, S.O. Function of polyphenol oxidase in higher plants, Physiol. Plant., 60, 106, 1984. 54. Montalbini, P., Buchanan, B.B., and Hutchesan, S.W. Effect of rust infection on rates of photochemical polychemical oxidation of latent polyphenol oxidase of Vicia faba chloroplast membranes, Physiol. Plant Pathol., 18, 51, 1981. 55. Takahama, U. Oxidation of vacuolar and apoplastic phenolic substrates by peroxidase: Physiological significance of the oxidation reactions, Phytochemistry, 3, 207, 2004. 56. Mayer, H.A. and Harel, E. Polyphenol oxidases in plants, Phytochemistry, 18, 193, 1979. 57. Tolbert, N.E. Activation of polyphenoloxidase of chloroplast, Plant Physiol., 51, 234, 1973. 58. Sherman, T.D., Gardeur, T.L., and Lax, A.R. Implications of the phylogenetic distribution of polyphenol oxidase in plants, in Enzymatic Browning and Its Prevention, Lee, C.Y. and Whitaker, J.R., Eds., American Chemical Society, Washington, DC, 1995, p. 103. 59. Chen, J.S. et al. Comparison of phenoloxidase activity from Florida spiny lobster and western Australian lobster, J. Food Sci., 56, 154, 1991. 60. Ali, M.T. et al. Activation mechanisms of pro-phenoloxidase on melanosis development in Florida spiny lobster and Western Australian lobster (Panulirus argus), J. Food Sci., 59, 1024, 1994. 61. Chevalier, T. et al. Molecular cloning and characterization of apricot fruit polyphenol oxidase, Plant Physiol., 119, 1261, 1999. 62. Yoruk, R. and Marshall, M.R. Physicochemical properties and function of plant polyphenol oxidase: A review, J. Food Chem., 27, 361, 2003. 63. Robinson, S.P., Loveys, B.R., and Chacko, E.K. Polyphenoloxidase enzymes in the sap and skin of mango fruit, Aust. J. Plant Physiol., 20, 99, 1993.

376


64. Kahn, V. Some biochemical properties of polyphenol oxidase from two avocado varieties differing in their browning rates, J. Food Sci., 42, 38, 1977. 65. Sanchez-Ferrer, A., Bru, R., and Garcia-Carmona, F. Novel procedure for extraction of a latent grape polyphenoloxidase using temperature-induced phase separation in Triton X-114, Plant Physiol., 91, 1481, 1989. 66. Richardson, T. and Hyslop, D.B. Enzymes, in Food Chemistry, 2nd ed., Fennema, O.R., Ed., Marcel Dekker, New York, 1985, p. 446. 67. Leydon Webb, J. Enzyme and Metabolic Inhibitors, vol. 2, Academic Press, New York, 1966, p. 301. 68. Walsh, C. Enzymatic Reaction Mechanism, W.H. Freeman & Company, San Francisco, CA, 1979, p. 461. 69. Espin, J.C. et al. Monophenolase activity of polyphenol oxidase from verdedoncella apple, J. Agric. Food Chem., 43, 2807, 1995. 70. Nicolas, J.J. et al. Enzymatic browning reactions in apple and apple products, Crit. Rev. Food Sci., 34, 109, 1994. 71. Marques, L., Fleuriet, A., and Macheix, J.J. Fruit polyphenol oxidases, new data on an old problem, in Enzymatic Browning and Its Prevention, Lee, C.Y. and Whitaker, J.R., Eds., American Chemical Society, Washington, DC, 1995, p. 90. 72. Wesche-Ebeling, P. and Montgomery, M.W. Strawberry polyphenol oxidase: Purification and characterization, J. Food Sci., 55, 1315, 1990. 73. Valero, E., Varon, R., and Garcia-Carmona, F. Characterization of polyphenoloxidase from Airen grapes, J. Food Sci., 53, 1482, 1988. 74. Oktay, M. et al. Polyphenol oxidase from Amasya apple, J. Food Sci., 60, 495, 1995. 75. Rocha, A.M.C.N. and Morais, A.M.M.B. Characterization of polyphenoloxidase (PPO) from “Jonagored” apple, Food Control, 12, 85, 2001. 76. Ngalani, J.A., Signoret, A., and Crouzet, J. Partial purification and properties of plantain polyphenoloxidase, Food Chem., 48, 341, 1993. 77. Park, E.Y. and Luh, B.S. Polyphenol oxidase of kiwifruit, J. Food Sci., 50, 678, 1985. 78. Galeazzi, M.A.M., Sgarbieri, V.C., and Constantinides, S.M. Isolation, purification and physicochemical characterization of polyphenoloxidases from a drawf variety of banana (musa cavendishii L.), J. Food Sci., 46, 150, 1981. 79. Tikoo, S. et al. Studies on polyphenol oxidase in wheat grains, Cereal Chem., 50, 520, 1973. 80. Halim, D.H. and Montgomery, M.W. Polyphenol oxidase of d’Anjou pears (pyrus communis L.), J. Food Sci., 43, 603, 1978. 81. Ratcliffee, B. et al. Tyrosinase, laccase and peroxidase in mushrooms (Agaricus, crimini, oyster, and shiitake), J. Food Sci., 59, 824, 1994. 82. Lerch, K. Copper monooxygenases, tyrosinases and dopamine-monooxygenase, in Metal Ions in Biological Systems. Copper Proteins, Siegel, H., Ed., Dekker, New York, 1981, p. 143. 83. Solomon, E.I. Copper proteins, in Metal Ions in Biology, Spiro, T.G., Ed., John Wiley & Sons, New York, 1981, p. 183. 84. Rescigno, A. et al. Tyrosinase inhibition: General and applied aspects, J. Enzyme Inhib. Med. Chem., 17, 207, 2002. 85. Solomon, E.I., Baldwin, M.J., and Lowrey, M.D. Electronic structures of active sites in copper proteins: Contributions to reactivity, Chem. Rev., 92, 521, 1992. 86. Whitaker, J.R. Polyphenol oxidase, in Food Enzymes Structure and Mechanism, Wong, D.W.S., Ed., Chapman & Hall, New York, 1995, p. 271. 87. Sánchez-Ferrer, A. et al. Tyrosinase: A comprehensive review of its mechanism, Biochim. Biophys. Acta, 1247, 1, 1995. 88. Decker, H. et al. Similar enzyme activation and catalysis in hemocyanins and tyrosinases, Gene, 398, 183, 2007. 89. Randhir, R., Lin, Y.-T., and Shetty, K. Phenolics, their antioxidant and antimicrobial activity in dark germinated fenugreek sprouts in response to peptide and phytochemical elicitors, Asia Pacific J. Clin. Nutr., 13, 295, 2004. 90. Bravo, L. Polyphenols: Chemistry, dietary sources, metabolism, and nutritional significance, Nutr. Rev., 56, 317, 1998. 91. Alasalvar, C. et al. Comparison of volatiles, phenolics, sugars, antioxidant vitamins, and sensory quality of different colored carrot varieties, J. Agric. Food Chem., 49, 1410, 2001. 92. Balasundram, N., Sundram, K., and Samman, S. Phenolic compounds in plants and agri-industrial by-products: Antioxidant activity, occurrence, and potential uses, Food Chem., 99, 191, 2006.


377

93. Wesche-Ebeling, P. and Montgomery, M.W. Strawberry polyphenoloxidase: Its role in anthocyanin degradation, J. Food Sci., 55, 731, 1990. 94. Martinez, M.V. and Whitaker, J.R. The biochemistry and control of enzymatic browning, Trends Food Sci. Technol., 6, 195, 1995. 95. Chutintrasri, B. and Noomhorm, A. Thermal inactivation of polyphenoloxidase in pineapple puree, Lebensm-Wiss-u-Technol., 39, 492, 2006. 96. Yemeniciog˘lu, A., Özkan, M., and Cemerog˘lu, B. Heat inactivation kinetics of apple polyphenoloxidase and activation of its latent form, J. Food Sci., 62, 508, 1997. 97. Khali, M. and Selselet-Attou, G. Effect of heat treatment on polyphenol oxidase and peroxidase activities in Algerian stored dates, Afr. J. Biotechnol., 6, 790, 2007. 98. Almeida, M.E. and Nogueira, J.N. The control of polyphenol oxidase activity in fruits and vegetables—A study of the interactions between the chemical compounds used and heat treatment, Plant Foods Hum. Nutr., 43, 245, 1995. 99. Lee, C.Y., Smith, N.L., and Hawbecker, D.E. Enzyme activity and quality of frozen green beans as affected by blanching and storage, J. Food Qual., 11, 279, 1988. 100. Ma, S.X. et al. Prevention of enzymatic darkening in frozen sweet potatoes [Ipomoea batatas (L.) Lam.] by water blanching: Relationship among darkening, phenols, and polyphenol oxidase activity, J. Agric. Food Chem., 40, 864, 1992. 101. Vina, S.Z. et al. Quality of Brussels sprouts (Brassica oleracea L. Gemmifera DC) as affected by blanching method, J. Food Eng., 80, 218, 2007. 102. Devece, C. et al. Enzyme inactivation analysis for industrial blanching applications: Comparison of microwave, conventional, and combination heat treatments on mushroom polyphenol oxidase activity, J. Agric. Food Chem., 47, 4506, 1999. 103. Flores, J.H. et al. Ultrafiltration of White Riesling juice: Effect of oxidation and pre-UF juice treatment on flux, composition, and stability, Am. J. Enol. Vitic., 39, 180, 1988. 104. Sims, C.A., Johnson, R.P., and Bates, R.P. Quality of a nonsulfited Vitis rotundifolia and a Euvitis hybrid white wine produced from ultrafiltered juice, Am. J. Enol. Vitic., 40, 272, 1989. 105. Goodwin, C.O. and Morris, J.R. Effect of ultrafiltration on wine quality and browning, Am. J. Enol. Vilic., 42, 347, 1991. 106. Drake, S.R. and Nelson, J.W. Apple juice quality as influenced by ultrafiltration, J. Food Qual., 9, 399, 1986. 107. Hsu, J.C. et al. Heat unstable proteins in grapejuice and wine. II. Characterization and removal by ultrafiltration, Am. J. Enol. Vitic., 38, 17, 1987. 108. Padilla, O.I. and McClellan M.R. Molecular weight cut-off of ultrafiltration membranes and the quality and stability of apple juice, J. Food Sci., 54, 1250, 1989. 109. Tanada-Palmu, P., Jardine, J., and Matta, V. Production of a banana (Musa cavendishii) extract containing no polyphenol oxidase by ultrafiltration, J. Sci. Food Agric., 79, 643, 1999. 110. Sims, C.A., Bates, R.P., and Arreola, A.G. Color, polyphenol oxidase, and sensory changes in banana juice as affected by heat and ultrafiltration, J. Food Qual., 17, 371, 1994. 111. Ludikhuyze, L.R. and Hendrickx, M.E. Effects of high pressure on chemical reactions related to food quality, in Ultra High Pressure Treatments of Foods, Hendrickx, M.E. and Knorr, D., Eds., Kluwer Academic/Plenum Publishers, New York, 2001, p. 167. 112. Seyderhelm, I. et al. Pressure induced inactivation of selected food enzymes, J. Food Sci., 61, 308, 1996. 113. Hendrickx, M. et al. Effects of high pressure on enzymes related to food quality, Trends Food Sci. Technol., 9, 197, 1998. 114. Ballestra, P. et al. Effect of high-pressure treatment on polyphenol oxidase activity of the Agaricus bisporus mushroom, High Press. Res., 22, 677, 2002. 115. Phunchaisri, C. and Apichartsrangkoon, A. Effects of ultra-high pressure on biochemical and physical modification of lychee (Litchi chinensis Sonn.), Food Chem., 93, 57, 2005. 116. Kim, Y.-S. et al. Effects of combined treatment of high hydrostatic pressure and mild heat on the quality of carrot juice, J. Food Sci., 66, 1355, 2001. 117. Hernandez, A. and Cano, M.P. High-pressure and temperature effects on enzyme inactivation in tomato puree, Agric. Food Chem., 46, 266, 1998. 118. Prestamo, G., Sanz, P.D., and Arroyo, G. Fruit preservation under high hydrostatic pressure, High Press. Res., 19, 535, 2000. 119. Guerrero-Beltran, J.A., Barbosa-Canovas, G.V., and Swanson, B.G. High hydrostatic pressure processing of peach puree with and without antibrowning agents, J. Food Process. Preserv., 28, 69, 2004.

378


120. Lopez, P. et al. Inactivation of peroxidase, lipoxygenase, and polyphenol oxidase by manothermosonication, J. Agric. Food Chem., 42, 252, 1994. 121. Vercet, A., Lopez, P., and Burgos, J. Inactivation of heat-resistant lipase and protease from Pseudomonas fluorescens by manothermosonication, J. Dairy Sci., 80, 29, 1997. 122. Vercet, A. et al. The effects of manothermosonication on tomato pectic enzymes and tomato paste rheological properties, J. Food Eng., 53, 273, 2002. 123. Christianson, D.D. et al. Supercritical fluid extraction of dry-milled corn germ with carbon dioxide, J. Food Sci., 49, 229, 1984. 124. Arreola, A.G. Effects of supercritical CO2 on some quality attributes of single strength orange juice, Masters thesis, University of Florida, Gainesville, FL, 1990. 125. Zemel, G.P. Low pH inactivation of polyphenoloxidase, Masters thesis, University of Florida, Gainesville, FL, 1989. 126. Chen, J.S. et al. Inactivation of polyphenol oxidase by high-pressure carbon dioxide, J. Agric. Food Chem., 40, 2345, 1992. 127. Gui, F. et al. Change of polyphenol oxidase activity, color, and browning degree during storage of cloudy apple juice treated by supercritical carbon dioxide, Eur. Food Res. Technol., 223, 427, 2006. 128. Sapers, G.M. Browning of foods: Control by sulfites, antioxidants, and other means, Food Technol., 47, 75, 1993. 129. McEvily, A.J., Iyengar, R., and Otwell, W.S. Inhibition of enzymatic browning in foods and beverages, Crit. Rev. Food Sci., 32, 253, 1992. 130. Rocha, A.M.C.N. and Morais, A.M.M.B. Polyphenol oxidase activity of minimally processed “Jonagord” apples (Malus domestica), J. Food Process. Preserv., 29, 8, 2005. 131. Manzocco, L., Mastrocola, D., and Poiana, M. Control of browning in frozen puree of cherimoya (Annona cherimola, Mill.) fruit, Sci. Des Aliments, 18, 101, 1998. 132. Dudley, E.D. and Hotchkiss, J.H. Cysteine as an inhibitor of polyphenol oxidase, J. Food Biochem., 13, 65, 1989. 133. ˙Iyidog˘an, N.F. and Bayındırlı, A. Effect of l-cysteine, kojic acid and 4-hexylresorcinol combination on inhibition of enzymatic browning in Amasya apple juice, J. Food Eng., 62, 299, 2004. 134. Dziezak, J.D. Preservatives: Antioxidants—The ultimate answer to oxidation, Food Technol., 40, 94, 1986. 135. Lambrecht, H.S. Sulfite substitutes for the prevention of enzymatic browning in foods, in Enzymatic Browning and Its Prevention, Lee, C.Y. and Whitaker, J.R., Eds., American Chemical Society, Washington, DC, 1995, p. 313. 136. Luo, Y. and Barbosa-Canovas, G.V. Enzymatic browning and its inhibition in new apple cultivars slices using 4-hexylresorcinol in combination with ascorbic acid, Food Sci. Technol. Int., 3, 195, 1997. 137. Guerrero-Beltran, J.A., Swanson, B.G., and Barbosa-Canovas, G.V. Inhibition of polyphenol oxidase in mango puree with 4-hexylresorcinol, cysteine and ascorbic acid, Lebensm-Wiss-u-Technol., 38, 625, 2005. 138. Walker, J.R.L. The control of enzymic browning in fruit juices by cinnamic acids, J. Food Technol., 11, 341, 1976. 139. Gunata, Y.Z., Sapis, J.C., and Moutonet, M. Substrates and aromatic carboxylic acid inhibitors of grape polyphenoloxidases, Phytochemistry, 26, 1573, 1987. 140. Kermasha, S., Goetghebeur, M., and Monfette, A. Studies on inhibition of mushroom polyphenol oxidase using chlorogenic acid as substrate, J. Agric. Food Chem., 41, 526, 1993. 141. Robert, C., Rouch, C., and Cadet, F. Inhibition of palmito (Acanthophoenix rubra) polyphenol oxidase by carboxylic acids, Food Chem., 59, 355,1997. 142. Jeon, M. and Zhao, Y. Honey in combination with vacuum impregnation to prevent enzymatic browning of fresh-cut apples, Int. J. Food Sci. Nutr., 56, 165, 2005. 143. Kim, M.-J., Kim, C.Y., and Park, I. Prevention of enzymatic browning of pear by onion extract, Food Chem., 89, 181, 2005. 144. Kim, C.Y. et al. Inhibition of polyphenol oxidase and peach juice browning by onion extract, Food Sci. Biotechnol., 16, 421, 2007. 145. Lozano-De-Gonzalez, P.G. et al. Enzymatic browning inhibited in fresh and dried apple rings by pinapple juice, J. Food Sci., 58, 399, 1993. 146. Lane, M.J. and Nursten, H.E. Variety of odors produced in Maillard model systems and how they are influenced by reaction conditions, in The Maillard Reaction in Foods and Nutrition, Waller, G.R. and Feather, M.S., Eds., ACS Symposium Series, vol. 215, American Chemical Society, Washington, DC, 1983, p. 141.


379

147. Muresan, S. et al. Aroma profile development of intermediate chocolate products. I. Volatile constituents of block-milk, Food Chem., 68, 167, 2000. 148. van Boekel, M.A.J.S. Formation of flavour compounds in the Maillard reaction, Biotech. Adv., 24, 230, 2006. 149. Matiacevich, S.B. and Buera, M.P. A critical evaluation of fluorescence as a potential marker for the Maillard reaction, Food Chem., 95, 423, 2006. 150. Knerr, T. et al. Formation of a novel colored product during the Maillard reaction of d-glucose, J. Agric. Food Chem., 49, 1966, 2001. 151. Feillet, P., Autran, J.C., and Icard-Verniere, C. Pasta brownness: An assessment, J. Cereal Sci., 32, 215, 2000. 152. Franzke, C. and Iwainsky, H. Antioxidant action of melanoidins, Deutsche Lebensmittel-Rundschau, 580, 251, 1954. 153. Morales, F.J. and Babbel, M.B. Antiradical efficiency of Maillard reaction mixtures in a hydrophilic media, J. Agric. Food Chem., 50, 2788, 2002. 154. Bersuder, P., Hole, M., and Smith, G. Antioxidants from a heated histidine-glucose model system. Investigation of the copper (II) binding ability, JAOCS, 78, 1079, 2001. 155. Ferrer, E. et al. Review: Indicators of damage of protein quality and nutritional value of milk, Food Sci. Technol. Int., 5, 447, 1999. 156. Nakamura, A. et al. Changes in allergenicity and digestibility of squid tropomyosin during the Maillard reaction with ribose, J. Agric. Food Chem., 54, 9529, 2006. 157. Erbersdobler, H.F. and Somoza, V. Forty years of furosine—Forty years of using Maillard reaction products as indicators of the nutritional quality of foods, Mol. Nutr. Food Res., 51, 423, 2007. 158. Nagao, M. et al. Mutagens in coffee and tea, Mutat. Res., 68, 101, 1979. 159. Kato, T., Hiramoto, K., and Kikugawa, K. Possible occurrence of new mutagens with the DNA breaking activity in coffee, Mutat. Res., 306, 9, 1994. 160. Swedish National Food Administration. Information about Acrylamide in Food, April 24, 2002. www.slv. se (accessed March 30, 2009). 161. Mottram, D.S., Wedzicha, B.I., and Dodson, A.T. Acrylamide is formed in the Maillard reaction, Nature, 419, 448, 2002. 162. Stadler, R.H. et al. Acrylamide from Maillard reaction products, Nature, 419, 449, 2002. 163. Becalski, A. et al. Acrylamide in foods: Occurrence, sources and modeling, J. Agric. Food Chem., 51, 802, 2003. 164. Taeymans, D. et al. A review of acrylamide: An industry perspective on research, analysis, formation and control, Crit. Rev. Food Sci., 44, 323, 2004. 165. Zhang, Y. and Zhang, Y. Formation and reduction of acrylamide in Maillard reaction: A review based on the current state of knowledge, Crit. Rev. Food Sci., 47, 521, 2007. 166. Prakash Reddy, V. et al. Involvement of Maillard reactions in Alzheimer disease, Neurotox. Res., 4, 191, 2002. 167. Tessier, F., Obrenovich, M., and Monnier, V.M. Structure and mechanism of formation of human lens fluorophore LM-1. Relationship to vesperlysine A and the advanced Maillard reaction in aging, diabetes, and cataractogenesis, J. Biol. Chem., 274, 20796, 1999. 168. Waanders, F. et al. Renal accumulation of pentosidine in non-diabetic proteinuria-induced renal damage in rats, Nephrol. Dial. Transpl., 20, 2060, 2005. 169. Hodge, J.E. Chemistry of browning reactions in model systems, J. Agric. Food Chem., 1, 928, 1953. 170. Hurst, D.T. Recent developments in the study of non-enzymic browning and its inhibition by sulphur dioxide, B.F.M.I.R.A. Scientific and Technical Surveys, No. 75, Leatherhead, England, U.K., 1972. 171. Kort, M.J. Reactions of free sugars with aqueous ammonia, Adv. Carbohydr. Chem. Biochem., 25, 311, 1970. 172. O’Brian, J. and Morrisey, P.A. Nutritional and toxicological aspects of the Maillard browning reaction in foods, Crit. Rev. Food Sci., 28, 211, 1989. 173. Kato, H. and Tsuchida, H. Estimation of melanoidin structure by pyrolysis and oxidation, Prog. Food Nutr. Sci., 5, 147, 1981. 174. Rubinsztain, Y. et al. Investigation on the structural units of melanoidins, Org. Geochem., 6, 797, 1984. 175. Blank, I. Current status of acrylamide research in food: Measurement, safety assessment, and formation, Ann. N. Y. Acad. Sci., 1043, 30, 2005. 176. Yaylayan, V.A., Wnorowski, A., and Locas, C.P. Why asparagine needs carbohydrates to generate acrylamide, J. Agric. Food Chem., 51, 1753, 2003.

380


177. Zyzak, D.V. et al. Acrylamide formation mechanism in heated foods, J. Agric. Food Chem., 51, 4782, 2003. 178. Granvogl, M. et al. Quantitation of 3-aminopropionamide in potatoes—A minor but potent precursor in acrylamide formation, J. Agric. Food Chem., 52, 4751, 2004. 179. Kwak, E.J. and Lim, S.I. The effect of sugar, amino acid, metal ion, and NaCl on model Maillard reaction under pH control, Amino Acids, 27, 85, 2004. 180. Miller, R., Olsson, K., and Pernemalm, P. Formation of aromatic compounds from carbohydrates. IX. Reaction of d-glucose and lysine in slightly acidic, aqueous solution, Acta Chem. Scand. B, 38, 689, 1984. 181. Wolfrom, M.L., Naoki, K., and Derek, H. Factors affecting the Maillard reaction between the sugars and amino acids studies on the nonenzymic browning of dehydrated orange juice, J. Agric. Food Chem., 22, 796, 1974. 182. Gög˘üs¸, F., Bozkurt, H., and Eren, S. Nonenzymic browning reactions in multi sugar and amino acid systems, J. Food Process. Preserv., 22, 81, 1998. 183. Massaro, S.A. and Labuza, T.P. Browning and amino acid loss in model total parental nutrition solutions, J. Food Sci., 55, 821, 1990. 184. Jousse, F. et al. Simplified kinetic scheme of flavour formation by the Maillard reaction, J. Food Sci., 67, 2534, 2002. 185. Brands, C.M.J., Wedzicha, B.L., and van Boekel, M.A.J.S. Quantification of melanoidin concentration in sugar–casein systems, J. Agric. Food Chem., 50, 1178, 2002. 186. Labuza, T.P. and Baisier, W.M. The kinetics of nonenzymatic browning, in Physical Chemistry of Foods, Schwarzberg, H.G. and Hartel, R.W., Eds., Marcel Dekker, Inc., New York, 1992, p. 595. 187. Hayashi, T. and Namiki, M. Role of sugar fragmentation in an early stage browning of amino-carbonyl reaction of sugar with amino acid, Agric. Biol. Chem., 50, 1965, 1986. 188. Pilkova, L., Pokorny, J., and Davidek, J. Browning reactions of Heyns rearrangement products, Nahrung, 34, 759, 1990. 189. Kato, K., Watanabe, K., and Sato, Y. Effect of the Maillard reaction on the attributes of ovalbumin, J. Agric. Food Chem., 29, 540, 1978. 190. Claeys, W.L., De Vleeschouwer, K., and Hendrickx, M.E. Kinetics of acrylamide formation and elimination during heating of an asparagine-sugar model system, J. Agric. Food Chem., 53, 9999, 2005. 191. Warmbier, H.C., Schnickels, R.A., and Labuza, T.P. Effect of glycerol on nonenzymatic browning in a solid intermediate moisture model food system, J. Food Sci., 20, 218, 1976. 192. Renn, P.T. and Sathe, S.K. Effects of pH, temperature, and reactant molar ratio on l-leucine and d-glucose Maillard browning reaction in an aqueous system, J. Agric. Food Chem., 45, 3782, 1997. 193. Kato, H., Yamamoto, M., and Fujimaki, M. Mechanisms of browning degradation of d-fructose in special comparison with d-glucose-glycine reaction, Agric. Biol. Chem., 33, 939, 1969. 194. Martins, S.I.F.S. and van Boekel, M.A.J.S. Kinetics of the glucose/glycine Maillard reaction pathways: Influences of pH and reactant initial concentrations, Food Chem., 92, 437, 2005. 195. Borsook, H. and Wasteneys, H. The interaction of free amino-nitrogen and glucose, Biochem. J., 19, 1028, 1925. 196. Huyghues-Despoints, A. and Yaylayan, V.A. Retro-aldol and redox reactions of Amadori compounds: Mechanistic studies with variously labelled D-[13 C] glucose, J. Agric. Food Chem., 44, 672, 1996. 197. Baxter, J.H. Free amino acid stability in reducing sugar systems, J. Food Sci., 60, 405, 1995. 198. Lertittikul, W., Benjakul, S., and Tanaka, M. Characteristics and antioxidative activity of Maillard reaction products from a porcine plasma protein-glucose model system as influenced by pH, Food Chem., 100, 669, 2007. 199. Rydberg, P. et al. Factors that influence the acrylamide content of heated foods, Chemistry and Safety of Acrylamide in Food Advances in Experimental Medicine and Biology, vol. 561, Springer Science + Business Media, Inc., New York, 2005, p. 317. 200. Benzing-Purdie, L.M., Ripmeester, J.A., and Ratcliffe, C.I. Effects of temperature on Maillard reaction products, J. Agric. Food Chem., 33, 31, 1985. 201. van Boekel, M.A.J.S. Kinetic aspects of the Maillard reaction: A critical review, Nahrung, 45, 150, 2001. 202. Miao, S. and Roos, Y.H. Nonenzymatic browning kinetics of a carbohydrate-based low-moisture food system at temperatures applicable to spray drying, J. Agric. Food Chem., 52, 5250, 2004. 203. Bozkurt, H., Gög˘üs¸, F., and Eren, S. Nonenzymic browning reactions in boiled grape juice and its models during storage, Food Chem., 64, 89, 1999.


381

204. Pedreschi, F., Kaack, K., and Granby, K. Acrylamide content and color development in fried potato strips, Food Res. Int., 39, 40, 2006. 205. Eichner, K. and Karel, M. The influence of water content and water activity on brown pigment formation, J. Agric. Food Chem., 20, 218, 1972. 206. Labuza, T.P., Tannenbaum, S.R., and Karel, M. Water content and stability of low moisture and intermediate moisture foods, Food Technol., 24, 543, 1970. 207. Eichner, K. The influence of water content on brown pigment formation in dehydrated foods and model systems and the inhibition of fat oxidation by browning intermediates, in Water Relations of Foods, Duckworth, R.B., Ed., Academic Press, New York, 1975, p. 417. 208. Gög˘üs¸, F., Düzdemir, C., and Eren, S. Effects of some hydrocolloids and water activity on nonenzymic browning of concentrated orange juice, Nahrung, 44, 438, 2000. 209. Ameur, L.A., Mathieu, O., and Lalanne, V. Comparison of the effects of sucrose and hexose on furfural formation and browning in cookies baked at different temperatures, Food Chem., 101, 1407, 2007. 210. Mundt, S. and Wedzicha, B.L. A kinetic model for browning in the baking of biscuits: Effects of water activity and temperature, Lebensm-Wiss-u-Technol., 40, 1078, 2007. 211. Sherwin, C.P. and Labuza, T.P. Role of moisture in Maillard browning reaction rate in intermediate moisture foods: Comparing solvent phase and matrix properties, J. Food Sci., 68, 588, 2003. 212. Jaiswal, A.K., Shukla, R., and Gupta, G. Control of Maillard browning in potato chips by yeast prefermentation, J. Food Sci. Technol. Mysore, 40, 546, 2003. 213. Rais, A. and Aroujalian, A. Reduction of the glucose syrup browning rate by the use of modified atmosphere packaging, J. Food Eng., 80, 370, 2007. 214. Moreno, F.J. et al. High-pressure effects on Maillard reaction between glucose and lysine, J. Agric. Food Chem., 51, 394, 2003. 215. Komthong, P. et al. Effect of high hydrostatic pressure combined with pH and temperature on glucose/ fructose-leucine/lysine/glutamate browning reactions, J. Fac. Agric. Kyushu Univ., 48, 135, 2003. 216. Gokmen, V. and Senyuva, H.Z. Acrylamide formation is prevented by divalent cations during the Maillard reaction, Food Chem., 103, 196, 2007. 217. Edelstein, D. and Brownlee, M. Mechanistic studies of advanced glycosylation end product inhibition by aminoguanidine, Diabetes, 41, 26, 1992. 218. Ingles, D.L. The formation of sulphonic acids from the reaction of reducing sugars with sulphite, Aust. J. Chem., 15, 342, 1962. 219. Wedzicha, B.L. and Vakalis N. Kinetics of the sulphite-inhibited Maillard reaction: The effect of sulphite ion, Food Chem., 27, 259, 1988. 220. Colahan-Sederstrom, P.M. and Peterson, D.G. Inhibition of key aroma compound generated during ultrahigh-temperature processing of bovine milk via epicatechin addition, J. Agric. Food Chem., 53, 398, 2005. 221. Schamberger, G.P. and Labuza, T.P. Effect of green tea flavonoids on Maillard browning in UHT milk, Lebensm-Wiss-u-Technol., 40, 1410, 2007. 222. Lindsay, R.C. and Jang, S. Model systems for evaluating factors affecting acrylamide formation in deep fried foods, Chemistry and Safety of Acrylamide in Food Advances in Experimental Medicine and Biology, vol. 561, Springer Science + Business Media, Inc., New York, 2005, p. 329. 223. Fernandez, S., Kurppa, L., and Hyvonen, L. Content of acrylamide decreased in potato chips with addition of a proprietary flavoniod spice mix (Flavomare) in frying, Inn. Food Technol., 18, 24, 2003. 224. International Agency for Research on Cancer, IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Some Industrial Chemicals, Acrylamide, vol. 60, Lyon, France: IARC, 1994, p. 389. 225. Pedreschi, F. et al. Color kinetics and acrylamide formation in NaCl soaked potato chips, J. Food Eng., 79, 989, 2007. 226. Gokmen, V. and Senyuva, H.Z. Effects of some cations on the formation of acrylamide and furfurals in glucose-asparagine model system, Eur. Food Res. Technol., 225, 815, 2007. 227. Kwak, E.J. and Lim, S.I. Inhibition of browning by antibrowning agents and phenolic acids or cinnamic acid in the glucose-lysine model, J. Sci. Food Agric., 85, 1337, 2005. 228. Bryce, D.J. and Greenwood, C.T. Thermal degradation of starch, Staerke, 15, 166, 1963. 229. O’Beirne, D. Effect of pH on nonenzymatic browning during storage in apple juice concentrate prepared from bramley’s seedling apples, J. Food Sci., 51, 1073, 1986. 230. Myers, D.V. and Howell, J.C. Characterization and specification of caramel colours: An overview, Food Chem. Toxicol., 30, 356, 1992. 231. Bozkurt, H. Kinetics of color changes due to Maillard Reactions in model systems, Masters thesis, University of Gaziantep, Gaziantep, Turkey, 1996.

382


232. Fagerson, I.S. Thermal degradation of carbohydrates—A review, J. Agric. Food Chem., 17, 747, 1969. 233. Kitts, D.D. et al. Chemistry and genotoxicity of caramelized sucrose, Mol. Nutr. Food Res., 50, 1180, 2006. 234. Schulz, A. et al. Electrospray ionization mass spectrometric investigations of a-dicarbonyl compounds— Probing intermediates formed in the course of the nonenzymatic browning reaction of l-ascorbic acid, Int. J. Mass Spectrom., 262, 169, 2007. 235. Huelin, P.E. Studies on the anaerobic decomposition of ascorbic acid, Food Res., 15, 78, 1953. 236. Monsalve, G.A., Powers, J.A., and Leung, H.K. Browning of dehydro ascorbic acid and chlorogenic acid as a function of water activity, J. Food Sci., 55, 1425, 1990. 237. Yuan, J.P. and Chen, F. Degradation of ascorbic acid in aqueous solution, J. Agric. Food Chem., 46, 5078, 1998. 238. Shinoda, Y. et al. Browning of model orange juice solution: Factors affecting the formation of decomposition products, Biosci. Biotech. Biochem., 69, 2129, 2005.

Oxidation and 12 Lipid Control of Oxidation Sotirios Kiokias, Theodoros H. Varzakas, Ioannis S. Arvanitoyannis, and Athanasios E. Labropoulos CONTENTS 12.1 Types of Oxidation in Oil Model Systems ........................................................................... 384 12.1.1 Introduction .............................................................................................................. 384 12.1.2 Autoxidation ............................................................................................................. 384 12.1.3 Azo-Initiated Oxidation............................................................................................ 385 12.1.4 Photosensitized Oxidation ........................................................................................ 385 12.1.5 Metal Catalyzed Oxidation ....................................................................................... 385 12.1.6 Enzyme-Catalyzed Oxidation .................................................................................. 386 12.1.7 Decomposition of Lipid Hydroperoxides (“Off-Flavor” Oxidation Products) ......... 387 12.1.8 Lipid Oxidation in Food Emulsions ......................................................................... 387 12.2 Lipid Stability Measurements ............................................................................................... 388 12.2.1 Evaluation of Primary Oxidation Products (“Lipid Hydroperoxides”) .................... 388 12.2.1.1 Measurement of Peroxide Value (POV)..................................................... 388 12.2.1.2 Determination of Lipid Hydroperoxides with the Ferric Thiocyanate Method ....................................................................................................... 388 12.2.1.3 Measurement of Conjugated Dienes (CD) ................................................. 388 12.2.2 Determination of Secondary Oxidation Products (“Off-Flavor” Volatiles) ............. 389 12.2.2.1 p-Anisidine Value Test ............................................................................... 389 12.2.2.2 Determination of Thiobarbituric Acid Related Substances ....................... 389 12.2.2.3 Determination of Volatile “Off-Flavor Products” with Gas Chromatography ........................................................................................ 390 12.2.3 Other Methods for the Evaluation of Oxidative Rancidity ....................................... 390 12.2.3.1 Measurement of Induction Time of Oxidation (Rancimat, Swift Test) ..... 390 12.2.3.2 Other Recent Oxidation Techniques .......................................................... 391 12.3 Control of Lipid Oxidation Vitamins as Natural Antioxidants ............................................ 391 12.3.1 General Information about Lipid Antioxidants ........................................................ 391 12.3.2 Carotenoids as Radical Scavengers and Oxygen Quenchers .................................... 393 12.3.3 Tocopherols (Vitamin-E) as Lipid Antioxidants ...................................................... 394 12.3.4 Activity of Ascorbic Acid (Vitamin-C) against Lipid Oxidation ............................. 396 12.3.5 Activity of Natural Antioxidant Mixtures against Lipid Oxidation ......................... 397 12.4 Legislation Related to Oil ..................................................................................................... 398 12.4.1 E.U. Legislation for Oils and Fats............................................................................. 398 12.4.2 U.S. Legislation for Oil ............................................................................................. 399 12.4.3 Canadian Legislation for Oil ....................................................................................402 References ......................................................................................................................................403 References (IT) ..............................................................................................................................408 383

384

12.1 12.1.1


TYPES OF OXIDATION IN OIL MODEL SYSTEMS INTRODUCTION

Free radicals (R•, ROO•, etc.) can be defined as any chemical species having one or more unpaired electrons, a wide definition that covers hydrogen atoms, transitions metals, the oxygen molecule itself, etc.1 One of the major areas in which carbon free radicals and oxygen free radicals are involved is lipid oxidation that determines the quality and shelf life of foods.2 Oxidative rancidity leads to the production of spoiled off-flavor products and is commonly associated with the organoleptic appraisal of food products.3 The oxidation of edible oils rich in polyunsaturated fatty acids is a major concern of the food industry because it is directly related to economic, nutritional, flavor, safety, and storage problems.4 Edible oils are susceptible to oxidation that produces undesirable volatile compounds and causes detrimental flavor effects in oil-based foods.5 The oxidation process is an important reaction occurring between unsaturated lipids and atmospheric oxygen and it is usually accelerated by the presence of metals, light, heat, and several initiators.6 Though lipid oxidation has been extensively studied in bulk oils, the relevant mechanisms have not been elucidated yet in lipid based emulsions.7 A better understanding of the factors monitoring the oxidative deterioration in these systems would offer strategies to improve the organoleptic and nutritional value of relevant food products.

12.1.2

AUTOXIDATION

Autoxidation is the oxidative deterioration of unsaturated fatty acids via an autocatalytic process consisting of a free radical mechanism. This indicates that the intermediates are radicals (R•, odd electron species) and that the reaction involves an initiation step and a propagation sequence, which continues until the operation of one or more termination steps.8 Autoxidation of lipid molecules is briefly described in the following reactions9: (I) Initiation RH ⇒ R• + H •

(Reaction 12.1)

or X• + RH ⇒ R• + XH

(Reaction 12.2)

R• + O2 ⇒ ROO•

(Reaction 12.3)

ROO• + RH ⇒ ROOH + R•

(Reaction 12.4)

R• + R• ⇒ R − R

(Reaction 12.5)

R• + ROO• ⇒ ROOR

(Reaction 12.6)

ROO• + ROO• ⇒ ROOR + O2

(Reaction 12.7)

(II) Propagation

(III) Termination

In the initiation step (Reactions 12.1 and 12.2), hydrogen is abstracted from an olefinic acid molecule (RH) to form alkyl radicals (R•), usually in the presence of a catalyst, such as metal ions, light, heat, or irradiation, at a relatively slow rate. The duration of the initiation stage varies for different lipids and depends on the degree of unsaturation and on the presence of natural antioxidants.10

Lipid Oxidation and Control of Oxidation

385

In the propagation sequence (Reactions 12.3 and 12.4), given an adequate supply of oxygen, the reaction between alkyl radicals and molecular oxygen is very fast and peroxyl radicals are formed (ROO•). These react with another fatty acid molecule producing hydroperoxides (ROOH) and new free radicals that contribute to the chain by reacting with another oxygen molecule. Hydroperoxide molecules can decompose in the presence of metals to produce alkoxyl radicals (RO •), which cleave into a complex mixture of aldehydes and other products, i.e., secondary oxidation products.11 The mutual annihilation of free radicals is known as the termination stage (Reactions 12.5 through 12.7), when the free radicals R• and ROO• interact to form stable, non-radical products. The rate of oxidation of fatty acids increases with their degree of unsaturation. The relative rate of autoxidation of oleate, linoleate, and linolenate is in the order of 1:40:100 on the basis of oxygen uptake and 1:12:25 on the basis of peroxide formation.12

12.1.3

AZO-INITIATED OXIDATION

An intrinsic problem during the investigation of lipid oxidative deterioration is the uncertainty about the rate of initiative reactions. One possible way of overcoming this problem is to introduce into the reaction mixture a compound that decomposes at a constant rate to free radicals (X•) capable of extracting a hydrogen atom from the fatty acid (RH) and consequently initiating the autoxidation process. The compounds most frequently used for this are the so-called azo-initiators13 (X−N=N=X) which thermally decompose to highly reactive carbon-centered radicals. Therefore, azo-initiators are useful for in vitro studies of lipid peroxidation generating free radicals spontaneously as following14: X − N = N − X ⇒ 2X • + N 2

(Reaction 12.8)

The water-soluble azo-initiator AAPH [2,2-azo-bis (2-amidinopropane) dihydrochloride] can be used to produce radicals in the aqueous phase, whereas the lipid-soluble AMVN [2,2’-azo-bis-(2,4dimethylvaleronitrile)] is commonly used to produce radicals in the lipid phase.15

12.1.4

PHOTOSENSITIZED OXIDATION

Photo-oxidation involves direct reaction of light-activated, singlet oxygen (1O2) with unsaturated fatty acids and the subsequent formation of hydroperoxides.16 In the most stable triplet state (two unpaired electrons in a magnetic field), oxygen is not very reactive with unsaturated compounds. Photosensitized oxidation involves reaction between a double bond and highly reactive singlet oxygen (paired electrons and no magnetic moment) produced from ordinary triplet oxygen by light in the presence of a sensitizer, such as chlorophyll, erythrosine, or methylene blue17 (Reactions 12.9 and 12.10) 1

sens + hν ⇒ 1sens* ⇒ 3sens* + 3O2

(Reaction 12.9)

sens* + 3O2 ⇒ 1sens + 1O2

(Reaction 12.10)

3

Singlet oxygen oxidation differs from autoxidation in several important respects: (1) It is an ene and not a radical chain reaction, (2) it gives products which are similar in type but not identical in structure to those obtained by autoxidation, and (3) it is a quicker reaction and its rate is related to the number of double bonds rather than the number of doubly activated allylic groups.10

12.1.5

METAL CATALYZED OXIDATION

Many natural oils contain metals such as cobalt, iron, magnesium, and copper, possessing two or more valence states with a suitable oxidation–reduction potential and serving thereby as excellent

386


prooxidants in lipid oxidation reactions.18 Contamination of oils with specific metals (copper, iron, etc.) can also occur during the refining procedure. Metals can initiate fatty acid oxidation by reaction with oxygen. The anion thus produced can either lose an electron to give singlet oxygen or react with a proton to form a peroxyl radical, which serves as a good chain initiator19 (Reaction 12.11).

Mn+ +O2

M(n+1)++O•– 2

1O2 (Reaction 12.11) •

OH

Many oxygenated complexes of transition metals have now been isolated and used as catalysts for oxidation of olefins, whereas recent evidence supports the initiation of autoxidation through the formation of a metal hydroperoxide catalyst complex.20 Once a small amount of hydroperoxides is formed, the transition metals can promote decomposition of the pre-formed hydroperoxides due to their unpaired electrons in 3d and 4d orbitals.21 A metal, capable of existing in two valence states typically acts as follows (Reactions 12.12 through 12.14): M n + + ROOH ⇒ RO• + OH − + M(n +1)+

(Reaction 12.12)

M(n +1)+ + ROOH ⇒ ROO• + H + + M n +

(Reaction 12.13)

2ROOH ⇒ RO* + ROO• + H 2O (Net reaction)

(Reaction 12.14)

In a system containing multivalent metal ions, such as Cu+ —Cu2+ or Fe2+ —Fe3+, the hydroperoxides can readily decompose to produce both RO • and ROO• as the metal ions undergo oxidation– reduction.22 Fukuzaka and Fujii23 reported that ferrous ions could catalyze the formation of alkoxyl radicals from linoleic acid hydroperoxides during oxidation of food emulsions. Chelation of metals by certain compounds decreases their prooxidant effect by reducing their redox potential and stabilizing the oxidizing form of the metal.4 A few natural acids (citric, phosphoric, tartaric, oxalic, etc.) and ethylenediamintetraacetic acid (EDTA) can chelate metals and thereby increase oxidation stability in oil model systems.9

12.1.6

ENZYME-CATALYZED OXIDATION

The basic chemistry of enzyme-catalyzed oxidation of food lipids such as in cereal products, or in many fruits and vegetables is the same as for autoxidation, but the enzyme lipoxygenase (LPX) is very specific for the substrate and for the method of oxidation.24 Lipoxygenases are globulins with molecular weights ranging from 0.6–1 × 105 Da, containing one iron atom per molecule at the active site. LPX type-1, can be found in many natural sources (e.g., potato, tomato, and soybean prefer polyunsaturated fatty acids), having as the best substrate linoleic acid (18:2), which is oxidized to 9 and 13 hydroperoxides.8 These hydroperoxides suffer fragmentation to give short-chain compounds (hexanal, 9-oxononanoic acid, 2-nonenal) some of which have marked and characteristic odors. LPX type-2 (present in gooseberry, soybean, and legumes) catalyzes oxidation of acylglycerols, whereas cooxidation of other plant components, e.g. carotenoids, may also occur.25 In that case, hydroperoxides may suffer enzyme-catalyzed reactions to give a mixture of products of oxidative deterioration as given in lipoxygenase enzymatic reaction (Reaction 12.15): ROH, RCHO, RH + O2

ROOH

ROOR, RCO2H

(Reaction 12.15)


12.1.7

387

DECOMPOSITION OF LIPID HYDROPEROXIDES (“OFF-FLAVOR” OXIDATION PRODUCTS)

A large body of scientific evidence suggests that the progressive loss of food palatability during lipid oxidation is due to the production of short chain compounds from the decomposition of lipid hydroperoxides.4,26 The volatile compounds produced from the oxidation of edible oils are influenced by the composition of the hydroperoxides and positions of oxidative cleavage of double bonds in the fatty acids.27 A variety of compounds such as hydrocarbons, alcohols, furans, aldehydes, ketones, and acid compounds are formed as secondary oxidation products and are responsible for the undesirable flavors and odors associated with rancid fat.28 The “off-flavor” properties of these compounds depend on the structure, concentration, threshold values, and the tested system. Aliphatic aldehydes are the most important volatile breakdown products because they are major contributors to unpleasant odors and flavors in food products.29 The peroxidation pathway from linoleic acid to various volatiles is determined in several researchs,7,30 by using various techniques (Gas chromatography mass spectrometry, GC–MS, and electron spin resonance spectroscopy, ESR), identified the volatile aldehydes that are produced during the oxidation of sunflower oil. In both cases, hexanal was the major aldehyde product of hydroperoxide decomposition, whereas pentanal, 2-heptenal, 2-octenal, 2-nonenal, 2,4-nonadienal, and 2,4-decadienal were also identified.

12.1.8

LIPID OXIDATION IN FOOD EMULSIONS

Because many common foods are emulsified materials (mayonnaise, coffee creamers, salad dressing, etc.), a better understanding of lipid oxidation mechanisms in emulsions is crucial for the formulation, production, and storage of these products.31,32 Moreover, apart from their technological importance, emulsion systems generally mimic the amphiphilic nature and the basic structural characteristics of important biological membranes (e.g., phospholipids), which are also prone to in vivo oxidative degradation when attacked by singlet oxygen and free radicals.33 In that aspect, in vitro research on the oxidative stability and antioxidation of model emulsions could provide with useful information of nutritional interest and thereby serve as pilot studies for in vivo clinical trials.34 Emulsions are thermodynamically unstable systems because of the positive energy required to increase the surface area between the oil and water phases.35 Generally, the stability of food emulsions is complex because it covers a large number of phenomena, including flocculation, coalescence, creaming, and final phase separation.36,37 Oil-in-water emulsions consist of three different components: water (the continuous phase), oil (the dispersed phase), and surface-active agents (emulsifiers at the interface). In such a system, the rate of oxidation is influenced by the emulsion composition (relative concentrations of substrate and emulsifier) and especially by the partition of the emulsifier between the interface and the water phase.38 Other factors influencing lipid oxidation in emulsions are particle size of the oil droplets, the ratio of oxidizable to non-oxidizable compounds in the emulsion droplets, and the packing properties of the surface-active molecules.39 In addition, the amount and composition of the oil phase in an emulsion are important factors that influence oxidative stability, formation of volatiles, and partition of the decomposition products, between the oil and water phase.40 A certain body of recent research has focused on the microstructural stability of protein stabilized oil-in-water emulsions that are structurally similar to recently developed foodstuffs (e.g., dairy alternative or “fresh cheese type” products, etc.).41–43 The image of such an emulsion has been visualized by the use of Confocal Laser Scanning Microscopy (CSLM).42 However, not much research has been done yet on the oxidative destabilization of these emulsion systems. A better understanding of the factors monitoring the oxidative deterioration of emulsions would offer antioxidant strategies to improve the organoleptic and nutritional value of the related products.

388

12.2 12.2.1


LIPID STABILITY MEASUREMENTS EVALUATION OF PRIMARY OXIDATION PRODUCTS (“LIPID HYDROPEROXIDES”)

12.2.1.1 Measurement of Peroxide Value (POV) One of the most commonly used methods for measuring rancidity is the peroxide value (POV), which is expressed in milliequivalents of oxygen per kilogram of fat or oil.44 This test is performed by iodometry, based on the reduction of hydroperoxide group (ROOH) with the iodide ion (I−). The concentration of the present peroxide is proportional to the amount of the released iodine (I2), which is assessed by titration against a standardized solution of sodium thiosulphate (Na2S2O3), using a starch indicator.45 (Reactions 12.16 and 12.17): 2ROOH + 2H + + 2KI → I 2 + 2ROH + H 2O + K 2O

(Reaction 12.16)

I 2 + 2Na 2S2O3 → Na 2S4O6 + 2NaI

(Reaction 12.17)

The iodometric method is rapid and applicable to all normal fats and oils. It is highly empirical however and results may be affected by the structure and reactivity of the peroxides as well as by the reaction temperature and time.46 Freshly refined fats and oils should have a POV value of 3 being the absolute maximum of the acceptable value, whereas it can be as high as 10 before any off-flavors that can be detected sensorically.9 12.2.1.2 Determination of Lipid Hydroperoxides with the Ferric Thiocyanate Method As an alternative approach to the determination of peroxide values, a colorimetric method has been reported in the literature.47 This method is based on the principle that the reduction of hydroperoxides can be accompanied by oxidation of Fe2+ to Fe3+ and determination of Fe3+ as ferric thiocyanate (Reactions 12.18 through 12.20). In more details, in the presence of hydroperoxides, the reaction of ferrous chloride with ammonium thiocyanate leads to the production of the red ferric thiocyanate chromophore that absorbs at 500 nm.8 Therefore, the higher the intensity of the chromophore the higher the amount of hydroperoxides in the sample. This method was found to be the most sensitive when compared with eight other photometric methods for evaluating fat deterioration.48 During the last years, there is an increasing application of the ferric thiocyanate method, in particular in experiments evaluating the lipid oxidation in emulsion systems.49 ROOH + Fe 2 + → RO• + Fe3+

(Reaction 12.18)

RO• + Fe 2 + + H + → ROH + Fe3+

(Reaction 12.19)

Fe3+ + 5SCN − → Fe (SCN )5

(Reaction 12.20)

2−

12.2.1.3 Measurement of Conjugated Dienes (CD) Oxidation of polyunsaturated fatty acids is accompanied by an increase in the ultraviolet absorption of the product. The spectra of the oxidized lipids are characterized by an intense absorption near 233 nm, with a lesser secondary absorption maximum due to ketone dienes in the region 260– 280 nm.50 The appearance of conjugated dienes in oxidized lipids is due to the double bond shift following free radical attack on hydrogens of methylene groups separating the double bonds in these compounds.51 Obviously, the determination of the conjugated dienes is a more sensitive oxidative indicator in linoleate (18:2) rich-substrates containing conjugated double bonds. In these lipid


389

systems, the amount of conjugated dienes can be quantified on the basis of absorbance at 232 nm and the molar absorptivity of linoleic acid, according to the following equation.52 Conjugated dienes (g/100 g of oil) =

1.0769*Abs232 Oil concentration in the sample (g/L)

Many authors have concluded that the conjugated diene method might be used as an index of stability of lipids in place of, or in addition to PV. It is faster than iodometric PV determination, much simpler, does not depend on chemical reactions or color development, and requires a smaller sample size.53 However, the presence of compounds absorbing in the region of the conjugated diene formation may interfere with such determinations. It has been proposed that interference in complex systems can be minimized or eliminated by derivative spectroscopy, using photodiode detection of spectra and computer analysis of the data.54

12.2.2 DETERMINATION OF SECONDARY OXIDATION PRODUCTS (“OFF-FLAVOR” VOLATILES) 12.2.2.1 p-Anisidine Value Test The extent of oxidation in fats and oils can be determined by the measurement of the formation of carbonyl compounds. The para-anisidine test determines the amount of aldehydes (principally 2-alkenals and 2,4-alkadienals) in vegetable and animal fats. Aldehydes in oil react with the p-anisidine reagent under acidic conditions to give yellowish products that absorb at 350 nm.55 The para-anisidine value (PAV) is defined as 100 times the absorbance of a solution resulting from the reaction of 1 g of fat or oil in 100 mL of a mixture of solvent and p-anisidine, measured at 350 nm in a 10 mm cell.56 A drawback of the method is that nonvolatile aldehydes also, for example 2,5 oxo-glycerides, can contribute to the absorption. As a rule of thumb, it can be said that good quality oil should have a PAV of less than 10.57 A good correlation between the p-anisidine value of salad oils and their organoleptic scores has been reported in the literature.58 The so-called TOTOX value has been used as an oxidative indicator,59 combining peroxide (POV) and p-anisidine values (PAV) as follows: TOTOX value = 2 (POV )+ PAV

(Reaction 12.21)

Although the totox value gives, somewhat a better, indication of the quality and rancidity of the oil, still it does always correlate well with sensorial observations, as it holds for both the POV and PAV values.60 12.2.2.2 Determination of Thiobarbituric Acid Related Substances The thiobarbituric acid (TBA) test is one of the most frequently used methods to assess lipid peroxidation, basically based on the determination of malonaldehyde which is assumed to be an important lipid oxidation product in food and biological systems.61 The reaction of malonaldehyde with the TBA reagent (Reaction 12.22) produces a pink complex with an absorption maximum at 532 nm.62 Therefore the increase in the amount of the producing red pigment as oxidative rancidity advances has been applied as a reliable oxidative indicator to a wide variety of foods. Several attempts have been made to establish a relationship between TBA values and the development of undesirable flavors in fats and oils.63 It has been shown that flavor threshold values correlate well with TBA results of vegetable oils such as those of soybean, corn, and safflower.64 However the chemical complexity of food and biological samples imposes certain limitations on the use of the TBA test for evaluating their oxidative state.44

390

Advances in Food Biochemistry Malonaldehyde

TBA HS

N

Red pigment HS

O è

O

O

+

2

HN

N

O

HN

NH O

OH

O

O

N H

(Reaction 12.22)

S

Production of TBAR chromophore (532 nm) during lipid oxidation

12.2.2.3 Determination of Volatile “Off-Flavor Products” with Gas Chromatography 12.2.2.3.1 Headspace Techniques (SPME Extraction) Various gas chromatographic (GC) methods, such as direct injection, dynamic headspace, and static headspace, have been used for the analysis of volatile products, resulting from the oxidative deterioration of vegetable oils.65 Though advantages and disadvantages are apparent with each GC method, for routine analyses, static headspace is the method of choice because it is rapid and requires no cleaning between samples.66 Solid-phase microextraction (SPME) is a versatile new sample preparation technique that has been used to measure the volatile products in lipid oxidation.67,68 In headspace SPME, there are two processes involved: the release of analytes from their matrix and the adsorption of analytes by the fiber coating.69 The volatile organic analytes are extracted, concentrated in the coating and transferred to the analytical instrument for desorption and analysis.70 In comparison to well-established techniques, SPME is inexpensive, solvent free, and convenient.71 In addition, because relatively mild conditions can be used, i.e., systems at equilibrium and temperatures less than 50°C, SPME gives a better quantitative estimate of the flavor profile.72 During the last years, appropriate SPME extraction techniques have been developed and successfully applied during oxidation of food related o/w emulsions in order to describe their volatile profile during oxidative deteroration.73 It has been found that the use of natural antioxidant mixtures has effectively inhibited the production of volatile aldehydes7,29 and thereby protect the final products from their organoleptic and nutritional deterioration. Kiokias and Oreopoulou7 described the profile of certain aldehydes, extracted with SPME during oxidation of sunflower o/w emulsions. 12.2.2.3.2 Non-Headspace Techniques In these techniques, the samples to be analyzed are directly introduced into the gas chromatic column.74 To maintain the quality of the column it is necessary to use a pre-column, which should be replaced regularly. However, a disadvantage of these techniques can be that primary oxidation products can react on the column to form additional values. Moreover, GC-sniffing techniques are applied to determine “off-flavor” compounds.75 With this method, a portion of the gas stream which leaves the column is led to the flame ionization detector while another part is used for sensorial determination.

12.2.3

OTHER METHODS FOR THE EVALUATION OF OXIDATIVE RANCIDITY

12.2.3.1 Measurement of Induction Time of Oxidation (Rancimat, Swift Test) Though it is scientifically accepted that lipid oxidation generally proceeds slowly in the initial stages, after a certain time (widely known as induction time) the oxidation rate starts increasing exponentially due to the fact that more unstable hydroperoxides are produced and subsequently are more easily broken down.4,27 Induction time is an important parameter for the quality of the lipid


391

substrate, dependent on many factors, such as type of oil, fatty acid composition, and presence of catalysts (e.g., metals, light, etc.).60 Several methods have been developed for measuring the induction period. The active oxygen test45 involves POV measurements of oil samples after bubbling of air in their interior and heating at 98°C. The Rancimat60,75 is an automated type of Swift Test, in which effluent gases after bubbling through the oil are led into a tube containing distilled water. During oxidation reactions various acids (e.g., formic, acetic, and propionic) are formed increasing the conductivity of the solution, which is recorded between two platinum electrodes. Drawbacks of these methods57 are that only bulk oils can be tested whereas volatile components may be lost during the oxidation process giving incorrect results for induction times. 12.2.3.2 Other Recent Oxidation Techniques Quite a few spectrophotometric techniques have been developed during the last years to estimate oxidative rancidity.76 Apart from the determination of conjugated diene hydroperoxides at 232 nm, the UV absorption at 268 nm has been commonly applied as a measure of volatiles due to the presence of unsaturated aldehydes absorbed in the region.77 Infrared spectroscopy has been used to follow the formation of trans double bonds. Moreover, chemi- and bioluminescence have also been used, based on the fact that the breakdown of the peroxides during oxidation is accompanied by the emission of light.4 Proton NMR has been used as a method to assess rancidity and to follow oxidation; in tested edible oils a good correlation between relative changes and TOTOX values has been established.53 Polarographic methods have been used, in which the oxidizing compounds are reduced at a dripping mercury electrode.78 Moreover with recently developed HPLC systems, it is in principle possible to identify various products of lipid oxidation.9 By using different types of columns and varying detection wavelengths a range of oxidizing products that cannot be analyzed by other techniques (such as alcohols and 2,5 glycerides, etc.) have been identified.79 For instance, Thin Layer Chromatography (TLC) has been used to rapidly assess the overall quality of oil.

12.3 12.3.1

CONTROL OF LIPID OXIDATION VITAMINS AS NATURAL ANTIOXIDANTS GENERAL INFORMATION ABOUT LIPID ANTIOXIDANTS

Lipid antioxidants can be broadly defined as any compounds serving to inhibit oxidative processes that cause deterioration of food lipids thereby improving the quality and extending the shelf life of the food products.80,81 It has been widely accepted that antioxidants for use in food systems must satisfy the following criteria82,83: 1. Being inexpensive, nontoxic, and effective at low concentrations 2. Having high stability and capability of surviving processing 3. Having no odor, taste, or color of its own but easy to incorporate and having a good solubility in the product Antioxidants may be added directly to the food system or as a solution in the food’s oil phase, in a food grade solvent or in an emulsified form that may be sprayed onto the food product.84 Based on their function, food antioxidants are classified as chain breaking or primary antioxidants, and synergists or secondary antioxidants.85 The primary antioxidants are free radical scavengers (FRS) that delay or inhibit the initiation step or interrupt the propagation step of autoxidation.86

392


They mainly donate hydrogen atoms to the lipid radicals (ROO* or RO*) to produce lipid derivatives and antioxidant radicals (FRS*) that are more stable and less readily available to promote autoxidation87 (Reaction 12.23). Chemical properties, including hydrogen bond energies, resonance delocalization, and susceptibility to oxidation, as well as the energy of the resulting free radical will influence the antioxidant effectiveness of the FRS.88 The antioxidant radical is stabilized by delocalization of the unpaired electron around a phenol ring to form stable resonance hybrids and finally participates in termination reactions resulting in non-radical dimers88 (Reaction 12.24). This means that each FRS is capable of inactivating at least two free radicals, the first being inactivated when the FRS interacts with the peroxyl radicals and the second when the FRS• enters a termination reaction with another peroxyl radical. This procedure can be pictured in (Reaction 12.25)

OH

ROO• or RO• + FRS ⇒ ROOH or ROH + FRS•

(Reaction 12.23)

FRS• + FRS• ⇒ FRS − FRS

(Reaction 12.24)

O*

O

O

OOR *

+ ROO*

(Reaction 12.25) Primary antioxidants are mono- or poly-hydroxy phenols with various ring substitutions including synthetic (BHA, BHT, PG), and natural compounds (tocopherols, carotenoids).89 Their antioxidant potency is determined by several factors, including chemical reactivity of the antioxidant toward the radical, concentration and mobility of the antioxidant in the microenvironment, and interaction with other antioxidants.90,91 Compounds that retard the rate of lipid oxidation by processes other than that of free radical scavenging are termed secondary antioxidants.92 Secondary antioxidants usually show antioxidant activity if a second minor component is present in the sample.93 Some of them are often called synergists because they promote the action of the primary antioxidants; citric acid, ascorbic acid, ascorbyl palmitate, lecithin, and tartaric acid are good examples of synergists.93 In the category of secondary antioxidants are also included compounds that bind metal ions (EDTA), reducing agents (e.g., ascorbic acid), and singlet oxygen quenchers (e.g., carotenoids).94 Regarding their origin, antioxidants are divided into synthetic and natural ones. The synthetic antioxidants mainly contain phenolic groups (such as butylated hydroxyanisole-BHA, propyl gallates-PG, etc.). Despite their superior efficacy and their high stability, there is increasing concern about the safety of synthetic antioxidants, including potential toxicity, allergenicity, etc.95,96 Thus the replacement of synthetic antioxidants by the “safer natural antioxidant” has been increasingly advocated, nowadays. Indeed, the food industry has a strong preference for the use of natural antioxidants, some of which may exist inherently in foods or be added intentionally during their processing.97,98 As shown by recent studies, a number of natural extracts from selected herbs (such as rosemary, sage, oregano, and thyme), which were found to be rich in polyphenols, flavonoids, and other compounds, have been well proved to be effective in retarding the development of rancidity in oils and fatty acids.99,100 Indeed, the activity of the natural extracts has been found to be dependent, among other factors, on the type and polarity of the extraction solvent, the isolation procedures, and the active components from the raw materials.101,102 The following sections focus on the antioxidant potential of several natural vitamins, such as carotenoids (provitamin-A), tocopherols (vitamin-E), and ascorbic acid (vitamin-C) against the oxidative deterioration of oil based systems. For each specific compound, the mechanisms of action


393

as well as the results of the relevant studies are reported, whereas some information on synergistic interactions between these vitamins is also recorded.

12.3.2

CAROTENOIDS AS RADICAL SCAVENGERS AND OXYGEN QUENCHERS

Carotenoids are a class of natural pigments, familiar to all through the orange-red to yellow colors of many fruits and vegetables as well as for the provitamin A activity that some of them possess.103 Most carotenoids are 40-carbon terpenoids having isoprene as their basic structural unit. A general subdivision is into “carotenes” which are strictly hydrocarbons (α- and β-carotene, lycopene) and “xanthophylls” which contain polar end groups reflecting an oxidative step in their formation (lutein, bixin, capsanthin, etc.).104 A large body of scientific evidence suggests that carotenoids scavenge and deactivate free radicals both in vitro and in vivo.105 It has been reported that their antioxidant action is determined by (1) electron transfer reactions and the stability of the antioxidant free radical; (2) the interplay with other antioxidants; and (3) their structure and the oxygen pressure of the microenvironment.103,104 Moreover, the antioxidant activity of carotenoids is characterized by literature data for (1) their relative rate of oxidation by a range of free radicals, or (2) their capacity to inhibit lipid peroxidation in multilamellar liposomes.106–109 According to Mordi110 the antioxidant activity of carotenoids is a direct consequence of the chemistry of their long polyene chain: a highly reactive, electron-rich system of conjugated double bonds susceptible to attack by electrophilic reagents, and forming stabilized radicals. Therefore, this structural feature is mainly responsible for the chemical reactivity of carotenoids toward oxidizing agents and free radicals, and consequently, for any antioxidant role.111 β-Carotene has received considerable attention in recent times as a putative chain breaking antioxidant, although it does not have the characteristic structural features associated with conventional primary antioxidants, but its ability to interact with free radicals including peroxyl radicals is well documented.112 Burton and Ingold113 were the first researchers who investigated the mechanisms by which β-carotene acts as a chain breaking antioxidant. In fact, the extensive system of conjugated double bonds makes carotenoids very susceptible to radical addition, a procedure which eventually leads to the free radical form of the carotenoid molecule. According to this particular mechanism, β-carotene is capable of scavenging peroxyl radicals (Reaction 12.26). The resulting carbon centered radical (ROO-β-CAR•) reacts rapidly and reversibly with oxygen to form a new, chain-carrying peroxyl radical (ROO-β-CAR-OO•). The carbon centered radical is resonance stabilized to such an extent that when the oxygen pressure is lowered the equilibrium of the Reaction 12.27 shifts sufficiently to the left, to effectively lower the concentration of the peroxyl radicals and hence reduces the amount of autoxidation in the system.114 Furthermore, the β-carotene radical adduct can also undergo termination by reaction with another peroxyl radical (Reactions 12.27 and 12.28). β -CAR + ROO• → ROO -β -CAR•

(Reaction 12.26)

ROO-β -CAR • + O2 ↔ ROO-β -CAR-OO•

(Reaction 12.27)

ROO-β -CAR • + ROO• → inactive products

(Reaction 12.28)

To understand the mechanism of antioxidant activity of the carotenoids it is also important to analyze the oxidation products that are formed during their action as antioxidants. A relationship between product-forming oxidation reactions to carotenoid antioxidant effects has been additionally proposed.115,116

394


In fact, in living organisms the partial pressure of oxygen in the capillaries of active muscle is only about 20 Torr, while in tissue it must be considerably lower.117 Certainly, this is consistent with the theory that carotenoids can operate as biological antioxidants under certain conditions, a theory that has been proved in many clinical trials.118,119 The carotenoid activity during oxidation is strongly influenced by the oxygen pressure (pO2) of the experimental conditions. Kiokias and Oreopoulou7 have shown that certain natural carotenoid mixtures (paprika, bixin and tomato, and palm-oil preparations) inhibited the azo-initiated oxidation of sunflower oil-in-water emulsions (operated rapidly under low pO2) in terms of both primary and secondary oxidation products. However, other studies120,121 concluded that carotenoids not only did not inhibit aerial lipid autoxidation (high pO2) but even exerted a prooxidant character, a phenomenon also observed at high carotenoid concentrations122 that could be due mainly to a more increased formation of carotene-peroxyl radicals, promoting the propagation of autoxidation. Interestingly, Kiokias8 observed that during aerial autoxidation of bulk and emulsified sunflower oil, various carotenoids strongly inhibited the production of volatile aldehydes, though they exerted no effect against the formation of hydroperoxides in the earlier stage of autoxidation, a finding that has been also reported by Warner and Frankel123 during autoxidation of soybean oil. The discovery that carotenoids deactivate singlet molecular oxygen was an important advance in understanding their technological and biological effects.124 According to Min and Bradley28 the addition of various carotenoids to foods containing unsaturated oils improves their shelf life. The mechanism by which carotenoids, and especially β-carotene which has been widely studied, act as oxygen quenchers can be summarized as follows94: In the presence of β-carotene, singlet oxygen will preferentially transfer exchange energy to produce the triplet state carotene, while oxygen comes back to its ground energy state to be inactivated (Reaction 12.29). Triplet state β-carotene releases energy in the form of heat, and the carotenoid is returned to its normal energy state (Reaction 12.30). In this way carotenoids act so effectively that one carotenoid molecule is able to quench up ∼1000 molecules of singlet oxygen. 1

O2 + β -carotene → 3 β -carotene* + 3O2

(Reaction 12.29)

β -carotene* → β -carotene + heat

(Reaction 12.30)

3

During chlorophyll sensitized photoxidation of edible oils, the carotenoid antioxidant effect was enhanced with an increasing concentration and number of double bonds.124–126 It has been reported that capsanthin which contains 11 conjugated double bonds, a conjugated keto-group, and a cyclopentane ring had higher antiphotooxidative activity than β-carotene, which has the same number of double bonds but neither of the functional groups.125,126 In a recent review paper by Kiokias and Gordon,127 the antioxidant activity of various carotenoids both in vitro and in vivo were summarized along with a reference of any reported prooxidant effects.

12.3.3 TOCOPHEROLS (VITAMIN-E) AS LIPID ANTIOXIDANTS Tocopherols and tocotrienols comprise a group of eight chromanol homologs that possess vitamin E activity in the diet. They are natural monophenolic compounds that can be found in plant tissues (e.g., nuts, vegetable oils, fruits, and vegetables).128 The tocopherols consist of a 6-chromanol group and an apolar phytyl chain, with the different forms (α-, β-, γ-, δ-) varying in the number and location of the methyl groups.129 The four tocotrienols are similar to corresponding tocopherols with the only difference being the unsaturation in the side chain at position 3′, 7′, and 11′. In vegetable oils, the tocopherol content depends very much on the growing conditions of the plant from which the oil is extracted as well as on the processing and storage conditions.4


395

In literature, due to differences in the used and tested systems there is still contradiction about the in vitro and in vivo antioxidant effects of tocopherol and tocotrienol isomers.130,131 Several factors have been reported to influence the tocopherol against lipid oxidation, such as 1. The rate by which tocopherols can transfer hydrogen atoms from the 6-hydroxyl group to the lipoperoxyl radical, which is ranked as following, between the different isomers: alpha > beta > gamma > delta. 2. Reactivity of the tocopheryl radical/stoichiometry that governs its conversion to the nonradical species. For instance, the sterically hindered α-tocopheryl radical is converted to a non-radical species slower than other isomers with δ-tocopherol being the fastest. 3. Stability of the tocopherols at elevated temperatures is in the following order: delta > gamma > beta > alpha. Taking into account that different orders of isomer activity have been previously reported, it can be concluded that antioxidant potential of tocopherols is mainly dependent on the tested lipid system. Indeed, the antioxidant activities of tocopherols have been investigated in various lipid substrates, including vegetable oils, animal fats, emulsions, PUFAs, etc.132 Huang et al.133 proposed that the relative antioxidant activity of different tocopherols depends on temperature, lipid composition, physical state (bulk phase, emulsion), and tocopherol concentration. In general, tocopherols behave as chain breaking antioxidants by competing with the fatty acids of their lipid substrate (RH) for the chain peroxyl radicals (ROO*).3 α-Tocopherols, for instance, donate a hydrogen to a peroxyl radical resulting in a α-tocopherol semiquinone radical (Reaction 12.31). This may further donate another hydrogen to produce methyl tocopherol quinone or react with another α-tocopheryl semiquinone radical (Reaction 12.32) to produce α-tocopherol dimer (Reaction 12.33) which also possesses antioxidant activity134: ROO• + α – toc ⇒ ROOH + α -toc–semiq •

(Reaction 12.31)

α -toc–semiq • + ROO• ⇒ ROOH and methyl–tocqin

(Reaction 12.32)

α -toc–semiq • + α -toc–semiq • ⇒ α -toc–dimer

(Reaction 12.33)

Several authors9,130 claimed that adding tocopherols to vegetable oils (even to the refined oils when a small tocopherol fraction was removed during deodorization) hardly shows any improvement in the oxidative stability due to the fact that an optimum concentration of these compounds is still present. In food manufacturing practice, it is recommended to keep the amount of the total α-tocopherol (natural or added) at levels between 50 and 500 ppm, depending on the kind of food product, because at sufficiently higher levels of addition the effect may become prooxidant.10 On the basis of hydroperoxide formation, α-tocopherol was found to exert an antioxidant activity at low concentrations but this changed to prooxidant effect at high concentrations (e.g. >1000 ppm).51 However, α-tocopherol still acted as an antioxidant with respect to the formation of volatiles, which is the determining factor in flavor keep-ability of oil- and fat-containing foods.135 α-Tocopherol is considered as the major antioxidant of olive oil with an activity dependent on both concentration and temperature.136 In aqueous linoleic acid micelles solution, α-tocopherol was a more effective antioxidant than γ–tocopherol.137 In another study, γ-tocopherol retained antioxidant activity at higher concentrations than α-tocopherol, though its effectiveness was not increased with concentration in soybean oil.138 Generally, it has been found that under common test conditions in oils, the antioxidant activity decreases from δ-tocopherol to α-tocopherol.139,140 However, several authors found that

396


δ-tocopherol acted as a better antioxidant than α-tocopherol, during autoxidation of linolate-rich substrates.129,141 It can be hypothesized that that the oxidation rate of samples containing tocopherols can be related to the number of methyl substituents on the chromanol ring, that would affect the stability of tocopherol and the tocopheroxyl radical, as well as the kinetics of hydrogen donation to a lipid radical.

12.3.4

ACTIVITY OF ASCORBIC ACID (VITAMIN-C) AGAINST LIPID OXIDATION

l-ascorbic acid, or vitamin C, is very widespread in nature, and it is increasingly gaining importance as a versatile natural food additive due both to its vitamin activity and its ability to improve the quality and extend the shelf life of many food products.142 Ascorbic acid is attractive as an antioxidant because as a food additive it has no usage limits and is highly recognized by the consumer as a nature-identical product.94 Vegetable foodstuffs (oranges, blackcurrants, parsley, green peppers, etc.) are the richest sources of vitamin C, but also animal products contain relatively smaller amounts. l-ascorbic acid is a six carbon weak acid with a pKa of 4.2, which is reversibly oxidized due to its enediol structure143 with the loss of an electron to form the free radical semihydroascorbic acid according to the Reaction 12.34. Activity of ascorbic acid as an antioxidant is shown in the ascorbic acid reaction (Reaction 12.34). Compared with other species, this radical is relatively stable and its further oxidation results in dehydroascorbic acid (DHASc) which probably exists in vivo in multiple CH2OH

CH2OH

CH2OH

CHOH O

CHOH O

CHOH O

–H

–e –H

O H

OH OH

CH2OH

O

CHOH O O

+e , +H

H

OH L

O

-Ascorbic anion

OH

O

Semi-dehydroascorbic radical

OO Dehydroascorbic acid

(Reaction 12.34) forms86, and can be reduced back to ascorbic acid by the same intermediate radical. In food systems, ascorbic acid is mainly a secondary antioxidant that can scavenge oxygen, act synergistically with chelators, and regenerate primary antioxidants. Indeed, depending on the conditions ascorbic acid can act through several mechanisms130,145,146: (1) hydrogen donation to regenerate the stable antioxidant radical; (2) metal inactivation to reduce the initiation of the metals; (3) hydroperoxide reduction to produce stable alcohols by non-radical processes; and (4) oxygen scavenging.99 In principle, ascorbic acid and its salts (sodium or calcium ascorbate) are water soluble antioxidants, not widely applicable for lipid systems but extensively used in beverages.128 In aqueous systems containing metals, ascorbic acid may also act as a prooxidant by reducing the metals that become active catalysts of oxidation in their lower valences. However, in the absence of added metals, ascorbic acid is an effective antioxidant at high concentrations.78 The action of ascorbic acid in lipid autoxidation is dependent on concentration, the presence of metal ions, and other antioxidants.47 It has been shown that ascorbates can protect plasma and LDL lipids from peroxidative damage, and it may inhibit the binding of copper ions to LDL.146 In several countries, ascorbic palmitate is used in fat containing foods due to its lipid solubility. However whether ascorbic palmitate exerts a better


397

activity than ascorbic acid is fully dependent on the tested system. Indeed, Cort147 observed that during oxidation of soya bean oil at 45°C, ascorbyl palmitate was much more effective than ascorbic acid, whereas exactly the opposite trend was found by Kiokias and Gordon29 in auto-oxidation of olive oil in water emulsions. Interestingly, in the latest research ascorbic acid was a superior antioxidant than its lipid homologue against the oxidative deterioration of olive oil.

12.3.5

ACTIVITY OF NATURAL ANTIOXIDANT MIXTURES AGAINST LIPID OXIDATION

Recent studies99,148,149 have shown that some natural antioxidants, such as vitamin E, vitamin C, and carotenoids can exhibit synergistic interactions, with the consequence that a combination of these compounds has a better antioxidant activity than the sum of the individual components. Mixtures of tocopherols and ascorbic acid have been reported to exhibit a synergistic effect during lipid oxidation. In a system containing both of these compounds, tocopherols are the primary free radical scavengers (FRS) because they are present in the lipid phase.150 The water soluble ascorbic acid can donate a hydrogen to tocopherols, so that tocopheroxyl radicals (TOC•), are reduced back to tocopherols (TOC), whereas ascorbic acid is converted to dehydroascorbic acid according to the Reaction 12.35: TOC• + Asc ⇒ TOC + DHAsc

(Reaction 12.35)

By this synergistic mechanism, tocopherols and ascorbic acid can mutually reinforce one another by regenerating their oxidized forms.151 Radical exchange reactions between lipid radicals, tocopherols, and ascorbic acid are the basis of numerous approaches for stabilizing oil and foods with their mixtures.97 Carotenoids were also reported to play a role in recycling phenolic antioxidants, e.g., tocopherols after one-electron oxidation.152 A recent laser photolysis study153 has shown that carotenoid radicals are reduced by α- or β-tocopherols via an electron transfer mechanism (Reactions 12.36 and 12.37). CAR • + TOH → CAR + TO•

(Reaction 12.36)

CAR + TO• → CAR • + TOH

(Reaction 12.37)

According to Bohm et al.154 a synergistic effect, observed in cell protection by β-carotene and vitamin E, may occur because it is not only quenching oxy-radicals but also repairing the α-tocopheroxyl radicals that are produced when α-tocopherol scavenges an oxy-radical. Such a synergistic mechanism requires that the CAR• is reconverted to CAR. However it has been also suggested that α-tocopherol protects β-carotene from being oxidized and not the other way around.114 Henry et al.121 found that a combination of β-carotene with α-tocopherol exhibited a stronger antioxidant effect than the individual antioxidants. Similarly, Lievonen155 found that a mixture of lutein with γ-tocopherol was strongly antioxidative while lutein with α-carotene had no effect on the oxidation of a purified triglyceride fraction from rapeseed oil. Kiokias and Gordon29 observed synergistic effects between norbixin and α-, δ-tocopherols against the production of “off-flavor” volatiles during the autoxidation of olive oil-in-water emulsions. Several in vitro studies have investigated interactions between carotenoids and vitamin C. It has been claimed154 that ascorbic acid can reduce the carotenoid radical cations in methanol according to Reaction 12.38: CAR •+ + AscH 2 → CAR + AscH • + H +

(Reaction 12.38)

398


According to Kanner156 the presence of an ascorbic acid–cupric ion couple at relatively high concentration inhibits carotene degradation in a β-carotene linoleate model system and thereby reinforces the antioxidant activity of the carotenoid. A tendency of increased carotenoid antioxidant activity in the presence of ascorbic acid has been observed during azo-initiated oxidation of sunflower oil-in-water emulsions.8 In the same oxidation system, an enhanced antioxidant activity of carotenoid mixtures (lutein, lycopene, paprika, bixin, etc.) have been reported7 as compared to each separate compound. Moreover, Kiokias and Gordon29 found that mixtures of olive oil phenolics with various carotenoids exhibited a strong activity against the autoxidation of bulk and emulsified olive oil, whereas individual carotenoids presented no inhibitory effect.

12.4 12.4.1

LEGISLATION RELATED TO OIL E.U. LEGISLATION FOR OILS AND FATS

According to E.U. Directive 96/3/EC (entry into force 17/2/1996) the bulk transport in sea-going vessels of liquid oils or fats that are to be processed, and that are intended for or likely to be used for human consumption, is permitted in tanks that are not exclusively reserved for the transport of foodstuffs, subject to the following conditions: I. That, where the oil or fat is transported in a stainless steel tank, or tank lined with epoxy resin or technical equivalent, the immediately previous cargo transported in the tank shall have been a foodstuff, or a cargo from the list of acceptable previous cargoes set out in the Annex, II. That, where the oil or fat is transported in a tank of materials other than those in point I, the three previous cargoes transported in the tanks shall have been foodstuffs, or from the list of acceptable previous cargoes set out in the Annex. The captain of the sea-going vessel transporting, in tanks, bulk liquid oils and fats intended for or likely to be used for human consumption shall keep accurate documentary evidence relating to the three previous cargoes carried in the tanks concerned, and the effectiveness of the cleaning process applied between these cargoes. Following Regulation (EEC) No. 136/66 (entry into force 1/10/1966) the Council shall fix a single production target price, a single market target price, a single intervention price, and a single threshold price for olive oil for the Community. Olive oil bought in by intervention agencies shall not be sold by them on the Community market on terms which might impede price formation at the level of the market target price. When olive oil is exported to third countries: (1) the difference between prices within the Community and prices on the world market may be covered by a refund where the former are higher than the latter and (2) a levy equal at most to the difference between prices on the world market and prices within the Community may be charged where the former are higher than the latter. Virgin olive oil produced by mechanical processes and free from any admixtures of other types of oil or of olive oil extracted in a different manner. Virgin olive oil is classified as follows: (a) Extra : olive oil of absolutely perfect flavor, with a free fatty acid content expressed as oleic acid of not more than 1 g per 100 g; (b) Fine : olive oil with the same characteristics as “Extra” but with a free fatty acid content expressed as oleic acid of not more than 1.75 g per 100 g; (c) Ordinary: olive oil of good flavor with a free fatty acid content expressed as oleic acid of not more than 3.73 g per 100 g; (d) Lampante: off-flavor olive oil or olive oil with a free fatty acid content expressed as oleic acid of more than 3.73 g per 100 g. Regulation (EEC) No 2568/91(entry into force 8/9/1991) makes clear that the characteristics of the oils shall be determined in accordance with the methods of analysis set out below: (a) for the determination of the free fatty acids, expressed as the percentage of oleic acid, (b) for the determination


399

of the peroxide index, (c) for the determination of aliphatic alcohols, (d) for the determination of the sterol content, (e) for the determination of erythrodiol and uvaol, (f) for the determination of the saturated fatty acids in position 2 of the triglyceride, (g) for the determination of the trilinolein content, (h) for spectrophotometric analysis, (i) for the determination of the fatty acid composition, (j) for the determination of the volatile halogenated solvents, (k) for the evaluation of the organoleptic characteristics of virgin olive oil, and (l) for proof that refining has taken place. Regulation (EC) No.2991/94 (entry into force 1/1/1996) laid down standards for: milk fats, fats, and fats composed of plant and/or animal products with a fat content of at least 10% but less than 90% by weight, intended for human consumption. The fat content excluding salt must be at least two-thirds of the dry matter. The Regulation applies also to products which remain solid at a temperature of 20°C, and which are suitable for use as spreads. The products may not be supplied or transferred without processing to the ultimate consumer either directly or through mass caterers, unless they meet the requirements set out. It has no application to the designation of products the exact nature of which is clear from traditional usage and/or when the designations are clearly used to describe a characteristic quality of the product and to concentrated products (butter, margarine, blends) with a fat content of 90% or more. The following information must be indicated in the labeling and presentation of the products: the sales description, the total percentage fat content by weight at the time of production for products, the vegetable, milk, or other animal fat content in decreasing order of weighted importance as a percentage by total weight at the time of production for compound fats and the percentage salt content must be indicated in a particularly legible manner in the list of ingredients. According to Regulation (EC) No.2815/98 (entry into force 31/10/2001) the designation of origin shall relate to a geographical area and may mention only: (a) a geographical area whose name has been registered as a protected designation of origin or protected geographical indication and/ or (b) for the purposes of this Regulation: a Member State, the European Community, a third country. The designation of origin, where this indicates the European Community or a Member State shall correspond to the geographical area in which the “extra virgin olive oil” or “virgin olive oil” was obtained. However, in the case of blends of “extra virgin olive oils” or “virgin olive oils” in which more than 75% originates in the same Member State or in the Community, the main origin may be designated provided that it is followed by the indication “selection of (extra) virgin olive oils more than 75% of which was obtained in…(designation of origin).” An extra virgin or virgin olive oil shall be deemed to have been obtained in a geographical area for the purposes of this paragraph only if that oil has been extracted from olives in a mill located within that area. Regulation (EC) No.1019/2002 (entry into force 1/11/2002) laid down specific standards for retail-stage marketing of the olive oils and olive-pomace oils. Oils shall be presented to the final consumer in packaging of a maximum capacity of 5 L. Such packaging shall be fitted with an opening system that can no longer be sealed after the fi rst time it is opened and shall be labeled. However, in the case of oils intended for consumption in mass caterers, the Member States may set a maximum capacity exceeding 5 L. Certain categories of oil are (a) extra virgin olive oil, (b) virgin olive oil, (c) olive oil composed of refined olive oils and virgin olive oils, and (d) olive-pomace oil. Only extra virgin and virgin olive oil may bear a designation of origin on the labeling. Some representative points and comments (repeals, modifications, amendments) of the E.U. Directive/ Regulations for oils–fats are given in Table 12.1.

12.4.2

U.S. LEGISLATION FOR OIL

According to International agreement on olive oil and table olives (Table 12.2) aims to the modernization of olive cultivation, olive oil extraction, and table olive processing:

400


TABLE 12.1 E.U. Directive and Regulations (Main Points and Comments) with Regard to Oils—Fats Directive—Title E.U. 96/3/EC (entry into force 17/2/1996)—Hygiene of foodstuffs as regards the transport of bulk liquid oils and fats by sea

Regulation No. 136/66/EEC (entry into force 1/10/1966)— Establishment of a common organization of the market in oils and fats

Main Points Definition of equivalent conditions to ensure the protection of public health and the safety and wholesomeness of the foodstuffs concerned. The bulk transport in sea-going vessels of liquid oils or fats is permitted in tanks that are not exclusively reserved for the transport of foodstuffs. Register of the three previous cargoes carried in the tanks concerned, and the effectiveness of the cleaning process applied between these cargoes. An export refund may be paid on exports of olive oil and rapeseed oil to cover the difference between the world and Community market prices.

Comments

Amendments—Regulation (EEC) No.1253/70 (entry into force 1/7/1970) No.1547/72 (entry into force 24/7/1972) No.1707/73 (entry into force 1/9/1973) No.2560/77 (entry into force 1/1/1978) No.1562/78 (entry into force 1/11/1978) No.1585/80(entry into force 27/6/1980) No. 3454/80 (entry into force 1/1/1980) No.1413/82 (entry into force 21/5/1982) No.2260/84 (entry into force 3/8/1984) No.231/85 (entry into force 3/2/1985) No.1454/86 (entry into force 24/5/1986) No.1915/87 (entry into force 3/7/1987) No.3994/87 (entry into force 1/1/1988) No.1098/88 (entry into force 29/4/1988) No.2210/88 (entry into force 26/7/1988) No.3499/90 (entry into force 5/12/1990) No.3577/90 (entry into force 1/1/1991) No.1720/91 (entry into force 26/6/1991) No.356/92 (entry into force 1/11/1992) No.2046/92 (entry into force 30/7/1992) No.3179/93 (entry into force 23/11/1993) No.3290/94 (entry into force 1/7/1995) No.1581/96 (entry into force 19/8/1996) No. 1638/98 (entry into force 4/8/1998) No.2702/1999 (entry into force 1/1/2000) No.2826/2000 (entry into force 1/1/2001)


401

TABLE 12.1 (continued) E.U. Directive and Regulations (Main Points and Comments) with Regard to Oils—Fats Directive—Title

Regulation (EEC) No 2568/91 (entry into force 8/9/1991)— The characteristics of olive oil and olive-residue oil and on the relevant methods of analysis

Regulation (EC) No.2991/94 (entry into force 1/1/1996)— Marketing standards for olive oil

Regulation (EC) No.2815/98 (entry into force 31/10/2001)— Marketing standards for olive oil

Regulation (EC) No.1019/2002 (entry into force 1/11/2002)— Standards for spreadable fats

Main Points Marketing standards, in particular covering labeling and quality grading, may be laid down for olive oil. Olive oil used for humanitarian aid purposes is purchased on the Community market or comes from intervention stocks. The characteristics of the oils shall be determined in accordance with the methods of analysis. The content of free fatty acids is expressed as acidity calculated conventionally. All the equipment used shall be free from reducing or oxidizing substances Application to milk fats and fats composed of plant and/or animal products. Not applicable to concentrated products. The products are supplied to the consumer only after processing. Products imported into the Community must comply with the Directive. The designation of origin shall relate to a geographical area. The “extra virgin olive oil” and “virgin olive oil” shall be packaged in an establishment approved for that purpose. Designation of origin checks in the packaging plants. Specific standards for retail-stage marketing of the olive oils and olive-pomace oils. Oils shall be presented to the final consumer in packaging of a maximum capacity of 5 L. Specific way of labeling

Comments Replacement and correction of articles

Amendment

Regulation (EC) No.1964/2002 (entry into force 1/7/2002) The products which were legally produced, were labeled and got into circulation before 1/1/2003 will be marketed until the consumption of the stocks.

402


1. To encourage research and development to elaborate techniques that could a. Modernize olive husbandry and the olive-products industry through technical and scientific planning b. Improve the quality of the products obtained therefrom c. Reduce the cost of production of the products obtained, particularly that olive oil, with a view to improving the position of that oil in the overall market for fluid edible vegetable oils d. Improve the situation of the olive products industry as regards the environment to abate any harmful effects 2. To encourage the transfer of technology and training in the olive sector “Olive oil” shall be restricted to oil obtained solely from the olive, to the exclusion of oil obtained by solvent or re-esterification processes and of any mixture with oils of other kinds. “Virgin olive oil” is oil which is obtained from the fruit of the olive tree solely by mechanical or other physical means under conditions, and particularly thermal conditions, that do not lead to deterioration of the oil, and which has not undergone any treatment other than washing, decantation, centrifugation, and filtration, to the exclusion of oil obtained by solvent or re-esterification processes and of any mixture with oils of other kinds. The main points of this agreement are given in Table 12.2.

12.4.3

CANADIAN LEGISLATION FOR OIL

For Edible oil products Act (Table 12.3) “edible oil product” means a food substance of whatever origin, source, or composition that is manufactured for human consumption wholly or in part from a fat or oil other than that of milk. No person shall manufacture or sell an edible oil product, other than oleomargarine, manufactured by any process by which fat or oil other than that of milk has been added to or mixed or blended with a dairy product in such manner that the resultant edible oil product is an imitation of or resembles a dairy product. The Lieutenant Governor in Council may make regulations (a) designating the edible oil products or classes of edible oil products to which this Act applies; (b) providing for the issue of licenses to manufacturers and wholesalers of any edible oil product and prescribing the form, terms, and conditions thereof and the fees to be paid therefor, and providing for the renewal, suspension, and cancellation thereof, (c) prescribing standards for the operation and maintenance of premises and facilities in which any edible oil product is manufactured, packed, or stored, (d) prescribing the standards of quality for and the composition of any edible oil product or class of edible oil product, (e) providing for the detention and confiscation of any edible oil product that does not comply with this Act and the regulations, (f) respecting the advertising of any edible oil product or class of edible oil product, (g) requiring and providing for the identification by labeling or otherwise of any edible oil product or class of edible oil product sold

TABLE 12.2 U.S. Agreement for Oil Title

Main Points

International agreement on olive oil and table olives, 1986

The objectives of this agreement were the international cooperation, the modernization of olive cultivation, olive oil extraction, and table olive processing. Definitions (olive oil crop year, olive products, etc.)

Comments Amendments

1993 (replacements and extension of the previous agreement) 2000 (prolongation of olive oil agreement)


403

TABLE 12.3 Canadian Act for Oil Title Edible oil products Act, 1990

Main Points Definitions (edible oil product, etc.) This Act applies to every edible oil product and class of edible oil product designated in the regulations.

Comments Amendments This Act is amended in 1994, 1999, and 2001.

or offered for sale, (h) prescribing the powers and duties of inspectors and analysts, (i) prescribing the records to be kept by manufacturers and wholesalers of any edible oil product, (j) exempting any manufacturer, wholesaler, or retailer of any edible oil product from this Act and the regulations, and prescribing terms and conditions therefor, (k) respecting any matter necessary or advisable to carry out effectively the intent and purpose of this Act. The main points of this Act are given in Table 12.3.

REFERENCES 1. Hamilton, R.J., Kalu, C., Prisk, E., Padley, B.F., and Pierce, H., Chemistry of free radicals in lipids, Food Chem., 60, 193, 1997. 2. Rice-Evans, A.C., Measurement of total antioxidant action as a marker of antioxidant status in vivo. Proceedings and limitations, Free Radical Res., 33, 59, 2000. 3. Frankel, E.N., Antioxidants in lipid foods and their impact on food quality, Food Chem., 57, 51–56, 1996. 4. Akoh, C.C. and Min, B.D., Food Lipid Chemistry, Nutrition and Biotechnology, Marcel Dekker, New York, 1997. 5. Gordon, M.H., Dietary antioxidants in disease, Nat. Product Rep., 13, 265, 1996. 6. Kiritsakis, A., Olive Oil-from Tree to the Table, 2nd ed., Food and Nutrition Press, Trumbull, CT, 1998. 7. Kiokias, S. and Oreopoulou, V., Antioxidant properties of natural carotenoid preparations against the AAPH-oxidation of food emulsions, Innov. Food Sci. Emerg. Tech., 7, 132–139, 2006. 8. Kiokias, S., In vitro and in vivo antioxidant properties of natural carotenoid mixtures. PhD thesis, School of Food Biosciences, The University of Reading, Reading, U.K., 2002. 9. Allen, J.C. and Hamilton, R.J., Rancidity in Foods, 3rd ed., Chapman & Hall, London, U.K., pp. 1–22, 1994. 10. Gunstone, F.D., Fatty Acid and Lipid Chemistry, Chapman & Hall, Glasgow, U.K., 1996. 11. Allen, J.C. and Hamilton, R.J., Rancidity in Foods, 3rd ed., Chapman & Hall, London, U.K., 1994. 12. Hsieh R.J. and Kinsella, E.J., Oxidation of PUFAs, mechanisms products and inhibition with emphasis on fish oil, Adv. Food. Nutr. Res., 33, 233, 1985. 13. Liegeois, C., Lernseau, G., and Collins, S., Measuring antioxidant efficiency of wort, malt, and hops against the 2,2,-azobis (2-qmidinopropane) dihydrochloride-induced oxidation of an aqueous dispersion of linoleic acid, J. Agric. Food. Chem., 48, 1129, 2000. 14. Niki, E., Free radical initiator as sources of water or lipid soluble peroxyl radicals, Methods Enzymol., 186, 100, 1990. 15. Esterbauer, H., The chemistry of oxidation of lipoproteins, in Oxidative Stress, Lipoproteins, and Cardiovascular Dysfunction, Rice-Evans, C. and Bruckdorfer, K.R. (Eds.), Portland Press, London, U.K., 1980. 16. Fakourelis, E.C. and Min, B.D., Effects of chlorophyll and β-carotene on the oxidation stability of olive oil, J. Food Sci., 52, 234, 1987. 17. David, W.R. and Dorr, A.L., Food Lipid Chemistry, Nutrition and Biotechnology, Akoh, C.C. and Min, B.D. (Eds.), Marcel Dekker, New York, 1997. 18. Dunford, H.B., Free radicals in iron-containing systems, Free Radical Biol. Med., 3, 405, 1987.

404


19. Mistry, B.S. and Min, B.D., Oxidised flavor compounds in edible oils. Developments in food science, in Off Flavours in Foods and Beverages, Charalambous, G. (Ed.), Elsevier, Amsterdam, the Netherlands, 1992. 20. Villiere, A., Vian, M., Brownec, I., Moreau, N., and Genot, C.J., Oxidative stability of bovine serum albumin- and sodium caseinate stabilised emulsions depends on metal availability, J. Agric. Food Chem., 53, 1514–1520, 2005. 21. Chen, J.H., Lee, T.C., and Ho, C.T. Antioxidant effect and kinetics study of capsanthin on the chlorophyll-sensitized photooxidation of soybean oil and selected flavor compounds, ACS Symp. Ser., 660, 188, 1997. 22. Mei, L. et al. Iron-catalysed lipid oxidation in emulsions as affected by surfactant, pH and NaCl, Food Chem., 61, 407, 1998. 23. Fukuzaka, K. and Fujii, T., Peroxide dependent and independent lipid peroxidation: Site specific mechanisms of initiation by chelated iron and inhibition by α-tocopherol, Lipids, 7, 227, 1998. 24 Yamamoto. S., Enzymatic lipid peroxidation, reactions of mammalians lipoxyganases, Free Radical Biol. Med., 10, 149, 1991. 25. Canfield, L.M., Bulux, J., and Serrano, J., Plasma response to oral β-carotene in Guatemala schoolchildren, Am. J. Clin. Nutr., 54, 539, 1991. 26. Aparicio, R. and Morales, M.T., Relationship between volatile compounds and sensory attributes of olive oil, J. Am. Oil. Chem. Soc., 73, 1253, 1996. 27. Frankel, E.N., Recent advances in lipid oxidation, J. Sci. Food. Agric., 54, 495, 1991. 28. Min, B.D. and Bradley, D.G., Fat and oil flavours, in Encyclopedia of Food Science & Technology, Wiley, New York, 1992. 29. Kiokias, S. and Gordon, M., Antioxidant properties of annatto carotenoids, Food Chem., 83, 523, 2003. 30. Keszler, A., Krisky, T., and Nenesh, A., Mechanisms of volatile compounds production during storage of sunflower oil, J. Agric. Food. Chem., 48, 1567, 2000. 31. Ponginelbi, I., Nawar, W.W., and Chinchoti, P., Oxidation of linoleic acid in emulsions. Effects of substrate, emulsifiers, and sugar concentration, J. Am. Oil Chem. Soc., 76, 131, 1999. 32. Kiokias, S., Lampa, K., Tsimogiannis, D., and Oreopoulou, V., Inhibition of oxidative deterioration in food emulsions, in Proccedings of INTRAFOOD-EFFoST Conference, Valencia, Spain, Vol. 2, p. 1237, 2005. 33. Halliwell, B. and Gutteridge, J., Free Radicals in Biology and Medicine, 2nd ed., Clarendon Press, Oxford, U.K., 1995. 34. Kiokias, S. and Gordon, M., Dietary supplementation with a natural carotenoid mixture decreases oxidative stress, Eur. J. Clin. Nutr., 57, 1135, 2003. 35. Dickinson, E., Introduction to Food Colloids, Oxford University Press, Oxford, U.K., 1997. 36. Friberg, E. and Larsson K., Food Emulsions, 3rd ed., Marcel Dekker, New York, 1996. 37. Kiokias, S., Reiffers-Magnani, C., and Bot, A., Stability of whey protein stabilized oil in water emulsions during chilled storage and temperature cycling. J. Agric. Food. Chem., 52, 3823, 2004. 38. Coupland, I.N. and Mc Clements, D.J., Lipid oxidation in food emulsions, Trends Food. Sci. Technol., 7, 83, 1996. 39. Labuza, T.P., Kinetics of lipid oxidation in foods, Crit. Rev. Food. Technol., 2, 355, 1971. 40. McClements, D.J. and Decker, E.A., J. Food Sci., 65, 1270, 2000. 41. Kiokias, S. and Bot, A., Temperature cycling stability of pre-heated acidified whey protein-stabilised o/w emulsion gels in relation to the internal surface area of the emulsion, Food Hydrocolloids, 20, 246, 2006. 42. Kiokias, S. and Bot, A., Effect of protein denaturation on temperature cycling stability of heat-treated acidified protein-stabilised o/w emulsions, Food Hydrocolloids, 19, 493, 2005. 43. Dickinson, E., Milk protein interfacial layers and the relationship to emulsion stability and rheology, Colloids Surf. B: Biointerfaces, 20, 197, 2001. 44. Moore, K. and Roberts, L.S., Measurement of lipid peroxidation, Radical Res., 28, 659, 1998. 45. Warner, K. and Eskin, N.M., Methods to Assess Quality and Stability of Oils and Fat Containing Foods, AOCS Press, Champaign, IL, 1995. 46. Gray, I.J., Measurement of lipid oxidation, J. Am. Oil Chem. Soc., 55, 539, 1978. 47. Ueda, S., Hayashi, T., and Namiki, M., Effect of ascorbic acid on lipid autoxidation in a model food system, Agric. Biol. Chem., 50, 1, 1986. 48. Tsoukalas, V. and Grosch, W., Analysis of food deterioration-Comparison of some photometric tests. J. Am. Oil Chem. Soc., 54, 490, 1977.


405

49. Hu, M., McClements, J., and Decker, E.A., Impact of whey protein emulsifiers on the oxidative stability of salmon oil-in-water emulsions, J. Agric. Food Chem., 51, 1435, 2003. 50. Logani, K.M. and Davis, R.E., Lipid oxidation: Biological effects and antioxidants, Lipids, 12, 485, 1980. 51. Huang, W., Frankel, E.N., Schwarz, K., and German, B., Effect of pH on antioxidant activity of α-tocopherol and trolox in oil-in-water emulsions, J. Agric. Food Chem., 42, 2496, 1996. 52. IUPAC, Standard Methods of Analysis of Oils Fats and Derivatives, 7th ed., Paquot, C. and Hautfenne, H. (Eds.), Blackwell Scientific Publications, Oxford, U.K., 1987. 53. Wanasundara, N.U., Shahidi, F., and Jablonski, C.R., Comparison of standard and NMR methodologies for assessment of oxidative stability of soybean oils, Food Chem., 52, 249, 1995. 54. Wheatley, A.R., Some recent trends in the analytical chemistry of lipid peroxidation, Trends Anal. Chem., 19, 620, 2000. 55. White, P.J., Conjugated diene, anisidine value and carbonyl value analysis, in Methods to Assess Quality and Stability of Oil and Fat Containing Foods, Warner, K. and Eskin, M. (Eds.), AOCS Publisher, Champaign, IL, p. 159, 1995. 56. Kasuga A., Aoyagi, Y., and Sugahara, T., J. Food Sci., 60, 1113, 1995. 57. Rossel, J.B., Rancidity in Foods, 3rd ed., Allen, J.C. and Hamilton, R.J. (Eds.), Blackie Academie & Professional, Glasgow, U.K., 1996. 58. List, G.R. et al., Oxidation and quality of soybean oil: A preliminary study of the anisidine test, J. Am. Oil Chem. Soc., 51, 17, 1974. 59. Osborn, H.T. and Akoh, C.C., Effect of emulsifier type, droplet size, and oil concentration on lipid oxidation in structured lipid-based oil-in-water emulsions, Food Chem., 84, 451, 2004. 60. Shaidi, F. and Wanasadura, U.N., Methods for measuring oxidative stability in fats and oils. In: Food Lipid Chemistry, Nutrition and Biotechnology, Akoh, C.C, and Min, B.D. (Eds.), Marcel Dekker, New York, 1997. 61. Coupland, J.N., Zhu, Z., Wan, H., McClements, D.J., Nawar, W.W., and Chinachoti, P., Droplet composition affects the rate of oxidation of emulsified ethyl linoleate, J. Am. Oil Chem. Soc., 73, 795, 1996. 62. Tarladgis, B.G., Pearson, A.M., and Dugan, R.L., Chemistry of the 2-TBA test for determination of oxidative stability in foods, J. Sci. Food. Agric., 15, 602, 1964. 63. McDonald, R.E. and Hultin, H.O., J. Food Sci., 52, 15, 1987. 64. Hawrysh, J.Z., Stability of conola oil, in Canola and Rapeseed Oil. Production, Chemistry, Nutrition, and Processing, Shahidi, F. (Ed.), Van Nortland Reinhlod, New York, p. 99, 1990. 65. Schieberle, P., Characteristion of Food: Emerging Methods, Gaonkar, A.G. (Ed.), Elsevier, London, U.K., 1995. 66. Snyder, J.M., Frankel, E.N., and Selke, E., Capillary gas chromatographic analysis of headspace volatiles from vegetable oils, J. Am. Oil Chem. Soc., 62, 1675, 1985. 67. Zabaras, D. and Wyllie, S.G., Re-arrangement of p-menthane terpenes by Carboxen during HS-SPME, J. Sep. Sci., 25, 685, 2002. 68. Steffen, A. and Pawliszyn, J., Analysis of flavor volatiles using headspace SPME, J. Agric. Food Chem., 92, 401, 1996. 69. Jimenez, A., Beltran, G., and Aguilera, M.P., Application of SPME to the analysis of volatile compounds in virgin olive oils, J. Agric. Food Chem., 46, 2744, 1998. 70. Brunton, P.N., Cronin, A.D., and Durkan, R., A comparison of SPME fibres for measurement of hexanal, and pentanal in cooked turkey, Food Chem., 68, 339, 2000. 71. Steenson, D.F., Lee, J.H., and Min, B.D., SPME microextraction of volatile soybean oil and corn oil compounds, J. Food Sci., 67, 71, 2002. 72. Yang, X. and Reppard, T., Solid phase microextraction for flavors analysis. J. Agric. Food Chem., 41, 1925, 1994. 73. Beltran, G., Aguilera, M.P., and Gordon, M.H., Solid phase microextraction of volatile oxidation compounds in oil-in-water emulsions, Food Chem., 92, 401, 2005. 74. Zegarska, Z. et al., Milchwissenchaft, 51, 195, 1996. 75. Karahadian, C. and Lindsay, R.C., Flavor Chemistry: Trends and Developments, Teranishi, R. et al. (Eds.), ACS, Washington, DC, 1989. 76. Slater, F.T., Overview of methods used for detecting lipid peroxidation, Methods Enzymol., 300, 283, 1995. 77. Oreopoulou, V., Extraction of natural antioxidants, in Extraction Optimization in Food Engineering, Tzia, C. and Liadiakis, G. (Eds.), Marcel Dekker, New York, pp. 329–346, 2003. 78. Porter, L.W., Black, D.E., and Drolet, M.A., Use of polyamide oxidative fluorescence test on lipid emulsions: Contrast in relative effectiveness of antioxidants in bulk versus dispersed systems, J. Agric. Food Chem., 37, 615, 1989.

406


79. Facino, R.M., Carini, M., Aldini, G., and Colombo, L., Characterization of the intermediate products of lipid peroxidation in phosphatidylcholine liposomes by fast-atom bombardment mass spectrometry and tandem mass spectrometry techniques, Rapid Commun. Mass Spec., 10, 1148, 1996. 80. Krinsky, N.I., Mechanism of actions of biological antioxidants, Proc. Soc. Exp. Biol. Med., 200, 298, 1992. 81. Loliger, J., The use of antioxidants in foods, in Free Radicals and Food Additives, Aruoma, I.O. and Haliwell, B. (Eds.), Taylor & Francis, London, U.K., 1990. 82. Fennema, O.R., Food Chemistry, Marcel Dekker, New York, 1996. 83. Kumpulainen, J.K. and Salonen, T.J., Natural Antioxidants and Food Quality in Atherosclerosis and Cancer Prevention, Royal Society of Chemistry, Cambridge, U.K., 1996. 84. Larson, R.A., Plant defenses against oxidative stress, Arch. Insect Biochem. Physiol., 29, 175, 2003. 85. Logani, M.K. and Davis, R.E., Lipid oxidation and effect of antioxidants, Lipids, 15, 485, 1981. 86. Buettner, G.R. and Moseley, P.L., EPR spin trapping of free radicals produced by bleomycin and ascorbate, Free Radical Res. Commun., 19, 589, 1993. 87. Cao, G. and Prior, R., Measurement of oxygen radical absorbance capacity in biological samples, Methods Enzymol., 299, 350, 1999. 88. Nawar, W.W., Lipids, in Food Chemistry, 3rd ed., Fennema, O. (Ed.), Dekker, New York, 1996. 89. Papadopulos, G. and Boskou, D., Antioxidant effect of natural phenols in olive oil, J. Am. Oil Chem. Soc., 68, 669, 1991. 90. Papas, M.A., Diet and antioxidant status, in Antioxidants in Human Health and Disease, Basu, K.T., Temple, J.N., and Gerg, L.M. (Eds.), CABI Publishing, New York, 1999. 91. Tsusihasci, H., Kigosi, M., and Niki, E., Action of β-carotene as an antioxidant against lipid peroxidation, Arch. Biochem. Biophys., 323, 137, 1995. 92. Gordon, M.H., The mechanism of antioxidant action in vitro, in Food Antioxidants, Hudson, F.J.B. (Eds.), Elsevier Applied Science, London, U.K., 1990. 93. Minotti, G., Sources and role of iron in lipid peroxidation, Chem. Res. Toxicol., 6, 134, 1993. 94. Reisch, W.D., Dorris, A., and Eitenmiller, R., Antioxidants, in Food Lipid Chemistry, Nutrition and Biotechnology, Akoh, C.C. and Min, B.D. (Eds.), Marcel Dekker, New York, 1997. 95. Hudson, B.J. and Lewis, J.I., Polyhydroxy flavonoid antioxidants for edible oils: Structural criteria for activity, Food Chem., 10, 47, 1983. 96. Thompson, M. and Moldens, P., Cytoxicity of BHA and BHT in rat hepatocytes, Biochem. Pharmacol., 37, 2201, 1983. 97. Loliger, J., Natural antioxidants, Lipid Technol., 3, 58, 1993. 98. Ramarathnam, N. et al., The contribution of plant food antioxidants to human health, Trends Food Sci. Technol., 6, 75, 1995. 99. Frankel, E.N. et al., Antioxidant activity of a rosemary extract and its constituents, carnosic acid, carnosol, and rosemarin acid, in bulk oil and oil-in-water emulsion, J. Agric. Food. Chem., 44, 131, 1996. 100. Takacsova, M., Pribela, A., and Faktorova, M., Study of the antioxidative effect of thyme, sage, juniper, and oregano, Die Nahung, 39, 241, 1995. 101. Abdalla, E.A. and Roozen, P.J., Effect of plant extracts on the oxidative stability of sunflower oil and emulsion, Food Chem., 64, 323, 1999. 102. Vekiari, S. et al., Oregano flavonoids as lipid antioxidants, J. Am. Oil Chemists Soc., 70, 483, 1993. 103. Packer, L., Antioxidant action of carotenoids-in vitro and in vivo protection against oxidation of human LDL, Ann. N. Y. Acad. Sci., 691, 48, 1993. 104. Khachik, F., Beecher, G.R., and Smith, J.C., Lutein, lycopene, and their oxidative metabolites in chemoprevention of cancer, J. Cell. Biochem., 22, 236, 1997. 105. Tessa. J. et al., Interactions between carotenoids and the CCl3O2 radical, Am. Chem. Soc., 117, 8322, 1995. 106. Bast, A., Haanen, G.R., and VandenBerg, H., Antioxidant effects of Carotenoids, Int. J. Vit. Nutr. Res., 68, 399, 1998. 107. Mortensen, A. and Skibsted, L.H., Importance of carotenoid structure in radical-scavenging reactions, J. Agric. Food. Chem., 45, 2970, 1997. 108. Mortensen, A. and Skibsted, L.H., Reactivity of β-carotene towards peroxyl radicals studied by later flash state photolysis, FEBS Lett., 426, 392, 1998. 109. Soffers, A.M.F., Boerma, M.G., Laanre, C., and Rietjens, I.M., Antioxidant activities of carotenoids: Quantitative relationships between theoretical calculations and experimental, Free Radical Res., 30, 233, 1999. 110. Mordi, R.C., Carotenoids-function and degradation, Chem. Industry, 110, 79, 1993.


407

111. Jorgensen, K. and Skibsted, L.H., Carotenoid scavenging of radicals—Effect of carotenoid structure and oxygen partial pressure on antioxidative activity, Zeitshrift furLebensmitteln, 196, 423, 1993. 112. Burton, W.G., Antioxidant action of carotenoids, Br. J. Nutr., 119, 109, 1988. 113. Burton, W.G and Ingold, K.U., Beta carotene: An unusual type of lipid antioxidant, Science, 224, 569, 1984. 114. Palozza, P. and Krinsky, N.I., Antioxidant effects of carotenoids in vitro and in vivo. An overview, Methods Enzymol., 213, 403, 1992. 115. Palozza, P., Calvello, G., and Bartoli, G.M., Prooxidant activity of β-carotene under 100% oxygen pressure in rat liver microsomes, Free Radical Biol. Med., 19, 887, 1995. 116. Liebler, D.C., Antioxidant reaction of carotenoids, Ann. N. Y. Acad. Sci., 691, 20, 1993. 117. Kasaikina, O.T. et al., Effect of environmental factors on the β-carotene reactivity toward oxygen and free radicals, Biol. Membr., 15, 168, 1998. 118. Olmedella, B., Granado, F., and Blanco, I., Supplementation with lutein and α-tocopherol in separate or combined oral doses in controlled men, Cancer Lett., 114, 179, 1997. 119. Dugas, T.R., Morel, D.W., and Harrison, E.H., Dietary supplementation with beta-carotene, but not with lycopene, inhibits endothelial cell mediated oxidation of LDL lipoprotein, Free Radical Biol. Med., 26, 1238, 1999. 120. Heinonen, M. et al., Inhibition of oxidation in 10% oil in water emulsions by β-carotene with α-, γ-, and δ-tocopherols, J. Am. Oil Chem. Soc., 74, 1047, 1997. 121. Henry, L.K., Gatignani, G.L., and Scwharz, S., The influence of carotenoids and tocopherols on the stability of safflower seed oil during heat-catalysed oxidation, J. Am. Oil Chem. Soc., 75, 1399, 1998. 122. Lominsky, S., Grossman, S., and Bergaman, H., In vitro and in vivo effects of β-carotene in rat epidermal lipoxygenase, Int. J. Vit. Res., 67, 407, 1997. 123. Warner, K. and Frankel, E.N., Effects of β-carotene on light stability of soyabean oil, J. Am. Oil Chem. Soc., 64, 213, 1987; Kiritsakis, A. and Dugan, L.R., Studies in photooxidation of olive oil, J. Am. Oil Chem. Soc., 62, 892, 1985. 124. Lee, H.S. and Min, B.D., Effects, quenching mechanisms, and kinetics of carotenoid in chlorophyllsensitized photo-oxidation of soyabean, J. Agric. Food Chem., 38, 1630, 1990. 125. Chen, J.H., Lee, T.C., and Ho, C.T., Antioxidant activities of caffeic acid and its related hydrocynamic acid compounds, J. Agric. Food Chem., 45, 2374, 1997. 126. Nielsen, B.R. et al., Singlet versus triplet reactivity in photodegradation of C40 carotenoids, J. Agric. Food Chem., 44, 2106, 1997. 127. Kiokias, S. and Gordon M., Properties of carotenoids in vitro and in vivo, Food Rev. Int., 20, 99, 2004. 128. Eitenmiller, R. and Laden, W.O., Vitamins, in Analyzing Food for Nutrition Labeling and Hazardous Contaminants, Jern, I.J. and Ikins, W.C. (Eds.), Dekker, New York, 1995. 129. Cillard, J. and Cillard, P., Behaviour of α-, γ-, and δ-tocopherols with linoleic acid in aqueous media, J. Am. Oil Chem. Soc., 57, 39, 1980. 130. Madhavi, L.D., Singhal, S.R., and Kulkavni, R.P., Technological aspects of foods antioxidants, in Food Antioxidants: Technological Toxicological and Health Perspectives, Madhavi, D.L. et al. (Eds.), Dekker, New York, pp. 159–266, 1996. 131. Schuler, P., Natural antioxidants exploited commercially, in Food Antioxidants, Hudson, F.B. (Ed.), Elsevier Applied Sciences, London, U.K., 1990. 132. Gottstein, T. and Gross, W., Model study of different antioxidant properties of α- and γ-tocopherols in fats, Food Sci. Technol., 92, 139, 1990. 133. Huang, W., Schwarz, K., German, B., Frankel E.N., and Hopia, A.I., Antioxidant activity of α-tocopherol and trolox in different lipids substrates: Bulk oil-in-water emulsions. J. Agric. Food Chem., 44, 444, 1996. 134. Kamal-Eldin, A. and Appelwist, L.A., The chemistry and antioxidant properties of tocopherols and tocotrienols, Lipids, 31, 671, 1996. 135. Frankel, E.N., Natural and biological antioxidants in food and biological systems. Their mechanisms of action, application and implications, Lipid Technol., 49, 77, 1995. 136. Marinova, E.M. and Yanislieva, N.V., Effect of temperature on antioxidant action of inhibitors in lipid autoxidation, J. Sci. Food. Agric., 60, 313, 1992. 137. Pryor, W.A., Cornicall, J.A., Dorall, L.I., and Tait, B., A rapid screening test to determine the antioxidant activity of natural and synthetic antioxidants, J. Org. Chem., 58, 3521, 1993. 138. Jung, M.Y. and Min, D.B., Effects of α-, γ-, and δ-tocopherols on oxidative stability of soybean oils, J. Food Sci., 55, 1464, 1992.

408


139. Lee, H.S. and Montag, A., Antioxidative wirksamkeit von tocochromanolen, Fett. Techn., 94, 213, 1992. 140. Hopia, A.I., Huang, S.W., Schwarz, K., German, B., and Frankel E.N., Effect of different lipid systems on antioxidant activity of rosemary constituents. Carnosol and carnosoic acid with and without α-tocopherol, J. Agric. Food Chem., 44, 2030, 1996. 141. Lee, H.S. and Wander, R.J., Relative activities of several tocopherols, J. Sci. Food Agric., 10, 537, 1959. 142. Coultate, T.P., Food—The Chemistry of Its Components, 2nd ed., The Royal Society of Chemistry, Cambridge, U.K., 1996. 143. Johnson, E.L., Food technology of the antioxidant nutrients, Crit. Rev. Food Sci. Nutr., 35, 149, 1995. 144. Counsel, J.N. and Horning D.H., Vitamin C, Ascorbic Acid, Applied Science, London, U.K., 1981. 145. Liao, M.T. and Seib, P.A., Selected reactions of L-ascorbic related to foods, Food Technol., 41, 104, 1987. 146. Stait, E.S. and Leake, S.D., Ascorbic acid can either increase or decrease LDL modification, FEBS Lett., 34, 263, 1994. 147. Cort, W.M., Antioxidant properties of ascorbic acid in foods, Adv. Chem. J., 200, 533, 1982. 148. Frankel, E.N., Huang, W., Kanner, J., and German, B., Interfacial phenomena in the evaluation of antioxidants: Bulk oils vs emulsions, J. Agric. Food Chem., 42, 1054, 1994. 149. Niki, E., Antioxidants in relation to lipid peroxidation, Chem. Phys. Lipids, 44, 227, 1987. 150. McCay, P.B., Vitamin E: Interactions with free radicals and ascorbate, Ann. Rev. Nutr., 5, 323, 1985. 151. Englard, S. and Seifter, S., The biochemical functions of ascorbic acid, Ann. Rev. Nutr., 6, 365, 1986. 152. Tan, B. and Saley, M.H., Antioxidant activities of tocopherols and tocotrienols on plant color carotenes, Am. Chem. Soc., 202, 39, 1991. 153. Mortensen, A. and Skibsted, L.H., Reactivity of β-carotene towards peroxyl radicals studied by later flash state photolysis, FEBS Lett., 426, 396, 1998. 154. Bohm, F., Edge, R., Lange, L., McGarvey, J., and Truscott, T.G., Carotenoids enhance vitamin E antioxidant activity, Am. Chem. Soc., 119, 621, 1997. 155. Lievonen, S., The effects of carotenoids on lipid oxidation. PhD Thesis. Department of Food Science, University of Helsinki, Helsinki, Finland, 1996. 156. Kanner, J., Pro-oxidant and antioxidant effects of ascorbic acid and metal salts in a β-carotene linoleate model system, J. Food Sci., 42, 60, 1977.

REFERENCES (IT) E.U. Directive and Regulations with regard to oils and fats E.U. 96/3/EC (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus!prod!DocNumber&1g=en&typ e_doc=Directive&an_doc=96&nu_doc=3) (accessed 20/03/2009). Regulation No. 136/66/EEC (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus!prod!DocNumber &1g=en&type_doc=Regulation&an_doc=66&nu_doc=136) (accessed 20/03/2009). Regulation (EEC) No 2568/91 (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus!prod!DocNumber &1g=en&type_doc=Regulation&an_doc=91&nu_doc=2568) (accessed 20/03/2009). Regulation (EC) No.2991/94 (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus!prod!DocNumber &1g=en&type_doc=Regulation&an_doc=94&nu_doc=2991) (accessed 20/03/2009). Regulation (EC) No.2815/98 (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus!prod!DocNumber &1g=en&type_doc=Regulation&an_doc=98&nu_doc=2815) (accessed 20/03/2009). Regulation (EC) No.1019/2002 (http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexplus!prod!DocNum ber&1g=en&type_doc=Regulation&an_doc=2002&nu_doc=1019) (accessed 20/03/2009). U.S. legislation related to oil http://r0.unctad.org/commodities/agreements/oliveen.pdf (accessed 20/03/2008). http://www.amazon.com/gp/product/9211122791/103-4320927-0003049?v=glance&n=283155 (accessed 20/03/2009). Canadian legislation for oil http://www.e-laws.gov.on.ca/DBLaws/RepealedStatutes/English/90e01_e.htm (accessed 20/03/2008). http://www.canlii.org/on/laws/sta/e-1/20041201/whole.html (accessed 20/03/2009).

Additives and 13 Food Contaminants Theodoros H. Varzakas, Ioannis S. Arvanitoyannis, and Athanasios E. Labropoulos CONTENTS 13.1 Introduction ......................................................................................................................... 410 13.2 Codex Committee on Food Additives and Contaminants ................................................... 411 13.3 Sweeteners—General Philosophy of Directive 94/35/EC on Sweeteners for Use in Foodstuffs............................................................................................................................411 13.3.1 Definition of Sweetener (FSA, (Regulation 2(1)) ................................................. 411 13.3.2 Permitted Sweeteners ............................................................................................ 412 13.3.3 Foods Allowed to Contain Permitted Sweeteners ................................................. 412 13.3.4 Sweeteners in Compound Foods—Carry-Over ..................................................... 412 13.3.5 Foods Not Allowed to Contain Sweeteners ........................................................... 412 13.3.6 Additional Labeling Requirements for Tabletop Sweeteners ................................ 413 13.4 Additives in Food Packaging .............................................................................................. 414 13.4.1 FDA Requirements ................................................................................................ 414 13.4.2 Synthetic/Vegetable-Derived Additives ................................................................. 414 13.4.3 Slips and Antistats ................................................................................................. 414 13.4.4 Antioxidants .......................................................................................................... 415 13.4.5 Colorants ............................................................................................................... 415 13.4.6 Antifogs ................................................................................................................. 416 13.4.7 Antimicrobials ....................................................................................................... 418 13.4.8 Freshness Indicators .............................................................................................. 418 13.4.9 Oxygen Scavengers ............................................................................................... 418 13.5 Additive Categories ............................................................................................................. 419 13.5.1 Acids ...................................................................................................................... 419 13.5.1.1 Lactic Acid ............................................................................................ 419 13.5.1.2 Succinic Acid ........................................................................................ 419 13.5.1.3 Fumaric Acid ........................................................................................ 420 13.5.1.4 Malic Acid ............................................................................................ 420 13.5.1.5 Tartaric Acid.......................................................................................... 421 13.5.1.6 Citric Acid ............................................................................................. 421 13.5.1.7 Ascorbic Acid........................................................................................ 421 13.5.1.8 Acetic Acid............................................................................................ 421 13.5.1.9 Sorbic Acid ........................................................................................... 421 13.5.1.10 Propionic Acid ...................................................................................... 422 13.5.1.11 Benzoic Acid ......................................................................................... 422 13.5.2 Coloring Agents..................................................................................................... 422

409

410


13.5.2.1 Dyes Requiring Certification for Their Use.......................................... 422 13.5.2.2 Dyes Not Requiring Certification for Their Use................................... 423 13.5.3 Aromatic Substances ............................................................................................. 426 13.5.3.1 Polycyclic Aromatic Hydrocarbons ...................................................... 427 13.5.3.2 Flavoring Substances ............................................................................ 428 13.5.4 Toxic Substances ................................................................................................... 429 13.5.4.1 Lead ...................................................................................................... 429 13.5.4.2 Mercury (Hg) ........................................................................................ 430 13.5.4.3 Cadmium (Cd) ...................................................................................... 431 13.5.4.4 Arsenic (As) .......................................................................................... 432 13.5.4.5 Selenium (Se)........................................................................................ 432 13.5.4.6 Antimonium (Sb) .................................................................................. 432 13.5.4.7 Aluminum (Al)...................................................................................... 433 13.5.4.8 Tin (Sn) ................................................................................................. 433 13.5.5 Medicinal Residues ............................................................................................... 433 13.5.6 Germicides............................................................................................................. 434 13.5.7 Preservatives .......................................................................................................... 435 13.5.7.1 Antimicrobial Substances ..................................................................... 435 13.5.7.2 Antioxidants .......................................................................................... 441 13.5.7.3 Antibiotics in Animal Feed ................................................................... 442 13.5.8 Packaging Materials .............................................................................................. 443 13.6 Toxicological Concerns....................................................................................................... 445 13.7 Do Additives Cause Health Problems? ............................................................................... 445 13.8 Risk Assessment of Food Additives .................................................................................... 450 References ...................................................................................................................................... 451

13.1

INTRODUCTION

Food additives have been used for centuries to improve food quality. Smoke, alcohols, and spices have been extensively used for the last 10,000 years as additives for food preservation. The abovementioned additives as well as a restricted number of additives comprised the main food additives until the Industrial Revolution. The Industrial Revolution brought so many changes in foods and asked for improved quality as well as quantity of the manufactured foods. For this reason many chemical substances were developed either for preservation or for color and/or odor enhancement. In the 1960s, over 2500 different chemical substances were used toward food manufacturing. In the United States over 2500 different additives were used to manufacture over 15,000 different foods. The desire for nutritional, functional, and tasty foods is an ongoing process. An additive is used to improve the shape, color, aroma, and extend the shelf life of a food. The following categories of additives are described: • • • • • • • •

Coloring agents (Tables 13.1 through 13.3) Acids (Table 13.5) Aromatic substances (Table 13.6) Toxic metals (Tables 13.9 and 13.10) Biocides Preservatives (Table 13.11) Emulsifiers (Table 13.13) Leavening agents (Table 13.14)

There has been an intense skepticism regarding the safe use of additives in foods. In the 1960s and 1970s the increase of toxicological information caused an increase in the knowledge of possible

Food Additives and Contaminants

411

risks derived from the consumption of foods containing additives. It was observed that the use of food additives has toxicological effects in humans. It is for this reason that in this chapter the limits of food additives use as well as pesticide residues’ control are mentioned. It is well known that there is a plethora of risks derived from additives, but also there are benefits from their use in food production. Additives will continue to play a significant role in food production since consumers continue to desire healthier, tastier, and occasionally functional foods and as the population of earth continues to increase.

13.2 CODEX COMMITTEE ON FOOD ADDITIVES AND CONTAMINANTS In the Codex Procedure Manual 13 edition (2003), it is clearly written that “All provisions in respect of food additives (including processing aids) and contaminants contained in Codex commodity standards should be referred to the Codex Committee on Food Additives and Contaminants preferably after the standards have been advanced to step 5 of the Procedure for the Elaboration of Codex Standards or before they are considered by the Commodity Committee concerned at Step 7, though such reference should not be allowed to delay the progress of the Standard to the subsequent Steps of the Procedure.” All provisions in respect of food additives will require to be endorsed by the Codex Committee on Food Additives and Contaminants, on the basis of technological justification submitted by the commodity committees and of the recommendations of the Joint FAO/WHO Expert Committee on Food Additives concerning the “safety-in-use (acceptable daily intake (ADI) and other restrictions) and an estimate of the potential and, where possible, the actual intake of the food additives, ensuring conformity with the General Principles for the Use of Food Additives”.

13.3

SWEETENERS—GENERAL PHILOSOPHY OF DIRECTIVE 94/35/EC ON SWEETENERS FOR USE IN FOODSTUFFS

For sweeteners to be included in this Directive, they first have to comply with the general criteria set out in Annex II of the Food Additives Framework Directive 89/107/EEC (OJL 40, 11,2,89, pp, 27–33). Under these criteria, food additives may only be approved if it has been demonstrated that they perform a useful purpose, are safe, and do not mislead the consumer, The recitals of the Directive 94/35/EC on sweeteners for use in foodstuffs further explain that the use of sweeteners to replace sugar is justified for the production of 1. Energy-reduced foods 2. Noncarcinogenic foods (i.e., foods that are unlikely to cause tooth decay) 3. Foods without added sugars, for the extension of shelf life through the replacement of sugar and for the production of dietetic products

13.3.1

DEFINITION OF SWEETENER (FSA, (REGULATION 2(1))

For the purposes of these regulations, a sweetener is defined as a food additive that is used or intended to be used either to impart a sweet taste to food or as a tabletop sweetener. Tabletop sweeteners are products that consist of, or include, any permitted sweeteners and are intended for sale to the ultimate consumer, normally for use as an alternative to sugar. Foods with sweetening properties, such as sugar and honey, are not additives and are excluded from the scope of this legislation. The Sweeteners in Food Regulations 1995 do not apply where a substance listed as a permitted sweetener is used for purposes other than sweetening, for example where sorbitol is used as a humectant in accordance with the Miscellaneous Food Additives Regulations 1995 and parallel Northern Ireland legislation.

412

13.3.2


PERMITTED SWEETENERS

(Regulations 2(1), 3(1), 4(a), and Schedule 1) The only sweeteners that are permitted for sale to the ultimate consumer or for use in or on food are those listed in Schedule 1 of the regulations whose specific purity criteria are in compliance with that stated in the annex to Directive 95/31/EC.

13.3.3 FOODS ALLOWED TO CONTAIN PERMITTED SWEETENERS (Regulations 3(2), 3(3), and Schedule 1) Permitted sweeteners are only allowed to be used in or on foods that fall within one of the categories listed in Schedule 1 of the regulations. A maximum usable dose for each permitted sweetener, varying according to the food category, is also specified within Schedule 1 and this must be respected. The use of two or more sweeteners in a single food is permitted, provided suitable categories exist and the maximum level for each individual sweetener is observed. The sale of foods that do not comply with these provisions is illegal.

13.3.4

SWEETENERS IN COMPOUND FOODS—CARRY-OVER

(Regulation 2(1) (amended 1997) and Regulation 5A) The regulations have been amended to include provisions on carry-over (Regulation 5A) to bring them into line with the GB Regulations on Colors and Miscellaneous Food Additives. These provisions allow the presence of a permitted sweetener in a compound food, to the extent that the sweetener is allowed by the regulations in one of the ingredients of the compound food. However, the definition of ‘compound foods’ in Regulation 2(1) means that permitted sweeteners are only allowed in the following compound foods: 1. Those with no added sugar or that are energy-reduced 2. Dietary foods intended for a low-calorie diet (excluding those specifically prepared for infants and young children) 3. Those with a long shelf life The regulations also provide for what is commonly known as “reverse carry-over.” This means permitted sweeteners can be present in foods (such as intermediary products) in which they would not otherwise be permitted, provided that these foods are to be used solely in the preparation of a compound food that will conform to the regulations.

13.3.5

FOODS NOT ALLOWED TO CONTAIN SWEETENERS

(Regulation 3(4) (amended 1997) and Regulation 5) The use of sweeteners in any foods for infants and young children is prohibited. This is specified in the Council Directive 89/398/EEC on the approximation of the laws of the member states relating to foodstuffs intended for particular nutritional uses (OJL 186, 30.6.89, pp. 27–32) and this prohibition now includes foods for infants and young children not in good health. The sale of such products containing sweeteners is also prohibited. Foods for infants and young children, generally known as “baby foods,” include foods specially prepared for infants and young children who are in good health; or whose digestive processes or metabolism is disturbed; or who have a special physiological condition where they would be able to obtain benefit from controlled consumption of certain substances in foods. For the purposes of this prohibition, Regulation 2(1) defines “infants” as children under the age of 12 months and “young children” as children aged between 1 and 3 years. These


413

definitions reflect those given in Article 1(2) of the Directive 91/321/EEC on infant formulae and follow-on formulae (OJL 175, 4.7.91, pp. 35–49) that are made under Directive 89/398/EEC. The terms “maximum usable dose” and “quantum satis” (Regulations 2(3)(c) and 2(3)(d)) These expressions are explained in the General Guidance Notes, Section 1, paragraphs 19 and 15 respectively. The terms “with no added sugar” and “energy-reduced” (Regulations 2(3)(a) and 2(3)(b)) Many of the categories listed in Schedule 1 of the regulations are described as “with no added sugar” or “energy-reduced.” The final product must comply with the definitions of these terms and the effect is to further restrict the type of foods in which sweeteners may be used. However, the actual terms “with no added sugar” or “energy-reduced” are not required by these Regulations to be used in the labeling of such products. Whatever description is used for those products must be in accordance with the Food Labeling Regulations 1996. “Energy-reduced” foods are foods with an energy value reduced by at least 30% compared with the original or a similar food. The legislation does not define the precise basis for this comparison, but wherever possible it should be by reference to one or more products that are currently on the market. If it is not possible to identify a comparable product that is currently on the market, the comparison could be made on the basis of previously marketed products. In an extreme case where it is not possible to identify an actual product, the comparison might be made with a hypothetically equivalent product, the composition of which is based on the use of sucrose rather than permitted sweeteners.

13.3.6

ADDITIONAL LABELING REQUIREMENTS FOR TABLETOP SWEETENERS

(Regulation 4(b)) The regulations include labeling requirements that apply to tabletop sweeteners only, In addition to the requirements contained within existing U.K. labeling legislation, tabletop sweeteners must include on their labels the phrase: “[Name of sweetener(s)]-based tabletop sweetener” Furthermore, where tabletop sweeteners contain polyols and/or aspartame, the following phrases must also be included on their labels: for polyols—“excessive consumption may induce laxative effects” for aspartame—“contains a source of phenylalanine” For the purposes of these Regulations, polyols are considered to be sorbitol and sorbitol syrup (E420 (i) and (ii)), mannitol (E421), isomalt (E953), maltitol and maltitol syrup (E965 (i) and (ii)), lactitol (E966), and xylitol (E967). The Additive and Food Contaminants (AFC) Panel of the European Food Safety Authority1 has evaluated the new long-term study on the carcinogenicity of aspartame conducted by the European Ramazzini Foundation (European Foundation of Oncology and Environmental Sciences) in Bologna, Italy. In its opinion, the Panel concluded, on the basis of all the evidence currently available, that there is neither need to further review the safety of aspartame nor to revise the previously established Acceptable Daily Intake (ADI) for aspartame (40 mg/kg body weight). The Panel also noted that intakes of aspartame in Europe, with levels up to 10 mg/kg body weight/day, are well below the ADI. Aspartame, an intense sweetener, has been authorized for use in foods and as a tabletop sweetener for more than 20 years in many countries throughout the world. Extensive investigations have been carried out on aspartame and its breakdown products through experimental animal and human studies, intake studies, and post-marketing surveillance.

414


In addition to a number of safety evaluations conducted in the past, the Scientific Committee on Food (SCF) carried out a review of all original and more recent studies on aspartame in 2002 and reconfirmed that aspartame is safe for human consumption.

13.4

ADDITIVES IN FOOD PACKAGING

13.4.1 FDA REQUIREMENTS Food packaging additives in the United States include slips, antistats, antioxidants, colorants, antifogs, antimicrobials, and oxygen scavengers. To obtain direct food contact approval, according to FDA requirements, materials must meet extractability requirements. The packaging material must not adulterate the food. A significant change in the FDA approval procedure was instituted in January 2000 with the new Food Contact Notification (FCN) system. To get approval for a new food-contact substance (FCS), the producer submits information including composition; intended use including additive level, usage temperature, and type of food the substance will contact; and data on migration of the substance into food. Migration studies can use food-simulating solvents, such as 10% ethanol to represent aqueous, acidic and low-alcohol foods, or food oil or 50% or 95% ethanol to represent fatty foods. Experimental temperature and duration are set at the most extreme anticipated conditions. For example, the most extreme condition requires heating at 121°C for 2 h, followed by 10 days at 40°C, in a special cell designed to withstand the extremes of temperature and pressure. The FDA uses migration data to estimate consumer exposure to the substance. In the previous system, an application could take several years to get approval. With the FCN system, the FDA has 120 days to review the application and object based on safety grounds, or the substance may be marketed. Expectations were that the new system would result in many more applications for new products from companies that otherwise would not have tried marketing in food applications.2 Although food companies have been driving a trend toward irradiation to prolong shelf life, general FDA guidance on package irradiation has not yet been published. Only a few polymers are approved for gamma irradiation of prepackaged food and these were approved with additive packages prevalent in the 1960s.

13.4.2

SYNTHETIC/VEGETABLE-DERIVED ADDITIVES

The trend continues toward use of synthetic or vegetable-oil-based rather than animal-fat-derived additives, agree industry experts. In Europe, this trend is due to the concern of bovine spongiform encephalopathy (BSE). Since many companies have customers globally, the concern has spread to the United States as well. Some companies continue to be interested in vegetable-based additives for kosher-certified applications.2

13.4.3

SLIPS AND ANTISTATS

Slip and antistat additives, which function at the surface of the plastic part, are traditionally migratory. The additives are difficult to predict and control because migration occurs over time and depends on part/film thickness and polymer crystallinity. Ampacet is a U.S. company that has introduced nonmigratory, surface-functional slip and antistat products that fit a need for controllable and predictable performance in premium films. Other advantages of the slip are that it can be used at higher temperatures than conventional slips and has no adverse effect on sealing. The nonmigratory antistat does not interact with adhesives, has no effect on sealing or printing, and has high thermal stability. Its antistatic properties last longer than those of conventional migrating antistats and its surface resistivity is similar at 50% and 12% relative humidity. The nonmigratory additives are used at much higher levels than traditional additives


415

and so are more expensive. They are extensively used in coextruded structures, such as a film that has slip on the inside but not on the outside. One trend for antistatic additives is the use of longer chain materials which provide higher temperature processing stability that is in increasing demand, as extruders are pushed to faster rates. A trend for slip additives is to use higher purity slips with reduced short chain (four- to nine-carbon) impurities.2 These higher purity slips are characterized by lower organoleptics, or taste and odor components. A company recently introduced a low organoleptic erucamide slip product called Incroslip that has advantages for the bottled water and beer industries. Plastic screw-type bottle closures contain high amounts of slip to enable torque release. Irradiation or sterilization of bottles by UV or ozone can degrade the trace amounts of by-products inherent in erucamide and produce off-tastes and odors.2

13.4.4 ANTIOXIDANTS High performance antioxidants are manufactured by chemical companies which perform well under harsh conditions such as gamma irradiation of food packaging. These usually have an improved solid phosphate or they are amine oxides derived from vegetable oils. Other products are phenolic antioxidants with reduced water extractability thus pertaining an advantage to liquid food products and to those applications requiring excellent post thermo-oxidative stability at 150°C after 14 days hot boiling water extraction. The antioxidant should not discolor, as typical phenolics do in either a quinone methide dimerization reaction in the dark that causes pinking or in a gas-staining reaction with prompt oxides of nitrogen that causes yellowing (Table 13.12).2 Vitamin E antioxidants are also used for improved organoleptic properties for sensitive applications such as plastic milk and beverage bottles. These are powerful stabilizers, effective at very low concentrations, because they react with carbon-centered radicals.

13.4.5

COLORANTS

A variety of organic and inorganic colorants are allowed by FDA for indirect food contact; other colorants are exempted from FDA regulation based on migration testing in a specific polymer for a specific application (Table 13.1). However, dyes are not allowed by FDA. The industry trend toward thinner parts (membranes, laminated films) creates a need for a higher colorant loading to maintain color intensity and opacity (Table 13.2). This has driven demand for

TABLE 13.1 Quantity of Coloring Agents Used in Certain Foods Food Categories

Dye Concentration (mg/kg)

Average Quantity (mg/kg)

Caramels-sweets Drinks Cereals Maraschino cherry Pet foods Ice creams Sausage Snack

10–400 5–200 200–500 100–400 100–400 10–200 40–250 25–500

100 75 350 200 200 30 125 200

Source: CCIC, Guidelines for good manufacturing practices: Use of certified FD&C colors in food, Certified Color Industry Committee, Food Technol., 22(8), 14, 1968.

416


TABLE 13.2 Representative Examples of Tint Arising from Mixing of Basic Dyes FD&C Blue No. 1

Tint Strawberry Black Egg white Cinnamon Green Green mint Orange Grape Black cherry Chocolate Caramel Peach Blackberry

FD&C Blue No. 2

FD&C Red No. 3 5

36

FD&C Red No. 40

FD&C Yellow No. 5

95 22

5 3 25

35

20 5 10 8 6

80 95 45 52

85 60 97 75

FD&C Yellow No. 6 42 15

100

21 60

5

75

45 40 64

9 40 20

Source: Adapted from CCIC, Food Technol., 22(8), 14, 1968.

higher pigment levels in “super concentrates” to maintain cost effectiveness. “High-efficiency” concentrates can contain pigment loadings of 75% or greater, compared to levels of 20%–50% for conventional concentrates (Table 13.3).2 The presence of flavored colorants (peach and raspberry), flavors (caramel, citric acid, and vanilla), and food preservatives (sodium nitrite, sodium nitrate, sodium benzoate, benzoic acid, potassium sorbate, and sodium chloride) in Escherichia coli suspension during exposure to sunlight was evaluated.3 He reported that these additives did not change the extent of cell survival. No effect on viability and mutation induction (kanamycin resistant) was also recorded when cells were in contact with any of the additives for 80 min in the dark. However, when the relevant additive was present in cell suspension during sunlight exposure the number of induced mutations was increased to varying extents over that seen with sunlight alone. Raspberry and peach increased the number of mutations in a dose-dependent manner, while vanilla produced mutations in an additive fashion. Nitrite, nitrate, benzoate, sorbate, and benzoic acid increased mutation somewhat additively over that of sunlight. Sodium chloride and citric acid were not effective. The impact of this investigation reflects the significance of the combination of sunlight and chemical food additives as potential risk, which requires special attention, and necessitates further investigations to evaluate the risk.

13.4.6

ANTIFOGS

In fresh-cut produce packaging, antifogs prevent the film from fogging so that the consumer can see the product clearly. The use of antifogs in fresh food packaging is on the increase and will continue into the future as new applications as well as new polymer entrants into the fresh food packaging industry continue to evolve.2 Antifogs act as a surfactant, so that moisture given off by produce forms a transparent, continuous film on the package surface rather than forming beads of water. Antifogs can be impregnated into the film as an additive or applied as a liquid coating. A new trend toward microwaving of fresh-cut produce packages, such as spinach products, has led to challenges of meeting performance requirements and regulatory requirements, which are stricter at elevated temperatures, producing films with


417

TABLE 13.3 Dyes Used for Food; Name, Origin, Functionality, Adverse Effects, ADI, and Applications E

Name

Origin

Functionality

E102

Tartrazine

Synthetic dye

Yellow color

E104

Cinolin yellow

Synthetic dye of coal tar

Cloudy yellow color

E107

Yellow 2G

Synthetic dye of coal tar

Yellow color

E120

Carmine

Red

E122

Azorubin

Natural color of extraction of dried insects Synthetic color

Red

E128

Red 2G

Synthetic dye

Red

E150 δ

Ammonium sulfite, caramel color

Synthetic dye derived from carbohydrates

Brown

E151

Bright black BN (boron nitride) or PN (brilliant black tetrasodium 4-acetamido5-hydroxy-6[7-sulfonato-4(4-sulfonatophenylazo)1-naphthylazo] naphthalene1,7-disulfonate)

Synthetic color of coal tar and nitrogen dye

Black

Effects

ADIa

Product Uses

Headaches in children, allergic reactions in adults Forbidden in Norway and Austria Low absorption from gastroenteric system Forbidden in Norway, United States, Australia, Japan Allergic reactions Forbidden in Norway, United States, Switzerland, Japan, Sweden Cancer

0–7.5

Fruit juices, cake mixtures, soups, ice creams, sauces, jams, yoghurt, sweets, gums, lollipops Smoked cod, ice creams

Allergic reactions to asthmatic people and those who are sensitive in aspirin, edema, retention of gastric juices In the intestine it gets transformed to aniline, which causes methemoglobinemia Gastroenteric problems, reduction in white blood cells of patients with B6 vitamin in low levels Intestine cysts in mice

0–0.5

—

0–2.5

Ice creams, alcoholic drinks

0–4.0

0–0.1

Sausages, cooked meat products, jams, drinks

—

Glucose tablets, ice cream, baking flour, total milled bread

0–1

Black sauces, chocolate mouse

Source: CFR, Code of Federal Regulations Title 21, Office of the Federal Register, U.S. Government Printing Office, Washington, DC, 1988. a ADI, acceptable daily intake mg/kg body weight.

418


an anti-fog coating. The coating also aids in improving the shelf life of the vegetables. The extended shelf life is due in part to the permeability of the package, but also to the “synergistic effect” of the antifog coating that reduces moisture, which can encourage the growth of spoilage bacteria.2

13.4.7 ANTIMICROBIALS The use of antimicrobials, or biocides, in packaging is a growing trend in the global food packaging industry. In the United States many of the antimicrobials in use protect the packaging or the packaging raw materials, although recent interest has been in antimicrobials to protect the packaged food. Antimicrobials that are incorporated into food packaging are regulated by the FDA under the Federal Food, Drug and Cosmetic Act (FFDCA). Under the FFDCA, the FDA ensures that such antimicrobial uses are safe with respect to any potential human dietary intake. Unrelated to federal requirements under the FFCDA, antimicrobial products used in food packaging that have no intended antimicrobial effect on the processed food in the package are subject to EPA registration as pesticides under the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA). Antimicrobials added to food or delivered to food via the packaging are treated as direct food additives and are not subject to FIFRA. Research at Clemson University has looked at coating food packages with nisin, particularly for hot dog packaging. Nisin can be compounded into the packaging polymer or applied as a powder or a coating. It is widely used in Europe but not extensively in the United States, although it has FDA approval.2 Silver compounds are also used in Europe and have FDA approval for some applications. Other research is looking at additives that produce the antimicrobial chlorine dioxide under certain relative humidity or UV light conditions. The advantage of these systems is that the antimicrobial could protect any product within the package, not just what comes in contact with a protective coating. Chlorine dioxide is also less expensive and effective for a broader range of microorganisms than nisin. The additives are currently being used in a sachet inside the package, but can be compounded into the packaging polymer.2

13.4.8

FRESHNESS INDICATORS

A major trend in the food industry over the past several years has been the consumer’s desire for freshness in taste and appearance and freshness indicators are an upcoming technology. Modified atmosphere packaging controls the flow of carbon dioxide through the food package to extend shelf life, but is dependent on storage conditions in the store or home. Indicators to show when a food has begun to decay are currently being used in bulk packaging, such as adhesive labels. Current research is focused on making indicators cost-effective for individual packaging. Indicator dyes work by either changing as a function of time and temperature or by reacting with a food degradation product. For example, an indicator in a sensor or in the packaging film could react with an amine given off by fish at the beginning of decay.2

13.4.9

OXYGEN SCAVENGERS

The use of oxygen absorbers is a relatively new additive trend in food packaging. Oxygen scavengers are especially important in the trend toward single-serve packages due to the smaller packages’ increased surface volume and exposure to oxygen. Commercial oxygen scavengers include iron oxide powders enclosed in sachets, additives incorporated into the packaging polymer or a polymer layer extruded as part of the package to maintain freshness by absorbing headspace oxygen and oxygen that enters the package. They can be incorporated directly into the walls of the package either as an existing layer within the package or as a distinct scavenging layer. The oxygen scavenging polymer system consists of an oxidizable resin, ethylene methyl acrylate cyclohexene methyl acrylate (EMCM) and a masterbatch containing a photoinitiator and a cobalt


419

salt catalyst. Other oxygen scavenging polymers based on nylon, polypropylene, polybutadiene, and polyisoprene degrade on oxidation into by-products that can migrate into packaged food and cause off-taste or odor. EMCM does not degrade into compounds responsible for off-taste or odor and the photoinitiator allows the inactive polymer to be stored and then activated by UV light during the package filling process.2 Cryovac introduced an improved oxygen scavenging film that reportedly removes oxygen 20% faster than before. The film slowed down microbial growth and oxidative deterioration of flavors, color, and nutrients. A Nestle fresh pasta package using the film recently won an award for technical innovation from the Flexible Packaging Association. The oxygen scavenging process increases the shelf life of refrigerated pasta.2

13.5 ADDITIVE CATEGORIES 13.5.1

ACIDS

13.5.1.1 Lactic Acid Dubos4 concluded that lactic acid has a bacteriostatic effect on Mycobacterium tuberculosis, which increased as pH decreased. Experiments carried out with Bacillus coagulans in tomato paste showed that lactic acid was four times more effective regarding the inhibition of bacterial growth compared to malic, citric, propionic, and acetic acid.5 Lactic acid is used in the jams, sweets, and drinks industries. This acid is the best acid to control the acidity and assure the transparency of brine in pickles.6 Calcium lactate can be used as a taste enhancer, as a baking processing aid, to proof the dough, as an inhibitor of decolorization of fruits and vegetables, as a gelatinization factor during pectins dehydration, or as an improver of the properties of milk powder or condensed milk.7 The ethyl esters of lactic acid can be used to enhance taste. Calcium lactate can be used in dietetic foods as well as nutrition supplements (Table 13.4). Lactic acid is an intermediate product of human metabolism. In the cases of pneumonia, tuberculosis, and heart failure, a non-physiological quantity of acid was detected in human blood. A growth problem was detected when acid was injected into water at a quantity of 40 mg/100 mL or in food at 45.6 mg/100 mL in hamsters. Lactic acid proved to be lethal in newborn fed milk with an unknown quantity of acid.8 The poisonous effect of acid has to do with its isomeric form. Babies fed with milk that has been acidified with D(−) or DL form suffered acidosis, lost weight, and dehydrated. Hence, only the L(+) form can be used in premature newborn babies. Acid is a food ingredient and an intermediate metabolite of human beings, therefore, there are no established limits of day consumption for humans.9 13.5.1.2 Succinic Acid FDA allowed the use of succinic acid as a taste enhancer and as a pH regulator.7 Succinic acid reacts with proteins and is used to modify the dough plasticity.6 Succinic acid derivatives can be used as taste enhancing agents or in combination with paraffins as a protective layer for fruits and vegetables. They can be used in pills production at a percentage not greater than 4.5%–5.5% of gelatin percentage and 15% of the total weight of capsule. Many derivatives of succinic acid are used as ingredients of paper and paperboard in food packaging. The succinic acid anhydrite is ideal for baking powder production of the Allied Chemical Co.6,10 The low level of acid hydrolysis is important during mixing of dough, since it is required that the additional acid should not react with soda during mixing until the product swells. In an acute toxicity study in rats with a daily subcutaneous injection of 0.5 mg for a period of 60 days, and with a dose increase of 2 mg/day during the fourth week, no detrimental effect was observed. Acid is produced in some fruits and constitutes an intermediate product of the cycle of Krebs. Hence, no established limits can be determined for humans.

420


TABLE 13.4 Maximum Amount of Organic Acids in Various Foods Foods Baked foods Drinks. nonalcoholic drinks Breakfast cereals Cheese Chewing gum Spices Dairy products Oils-fats Frozen dairy desserts Gelatin Sauces Hard candies Jam Meat products Fruit juices Snacks Soft candies

Acetic Acid

Calcium Acetate

0.25

0.2

Sodium Acetate

Sodium Diacetate

Adipic Acid

Caprilic Acid

0.4

0.05 0.005

0.013

Malic Acid

Succinic Acid

3.4

0.007 0.8 0.5 9.0 0.8 0.5

0.02

0.04 3.0

0.5

0.1

0.2

5.0 0.45 0.3 0.004 0.55

3.0

0.25 0.15 0.12 0.12

0.6

0.084 0.005 0.005 0.005 0.1

0.8 6.9

0.1

0.3

0.005

1.3

0.016 0.005

2.6 0.0061 3.5

0.6

0.05 0.2

0.1

3.0

Source: CFR, Code of Federal Regulations Title 21, Office of the Federal Register, U.S. Government Printing Office, Washington, DC, 1988.

13.5.1.3 Fumaric Acid Fumaric acid is responsible for a sour taste in foodstuffs. It is widely used in fruit juices, in desserts, in the frozen dough of biscuits, wild cherry liqueur, and in wines. It can also be used as a coating agent in caramels and bread. Fumaric acid contributes to the extension of the shelf life of baking powder due to its restricted solubility and the low humidity absorption. It attains very good antioxidant properties and is used to avoid the rancidity of pork fat, butter, milk powder, sausages, bacon, walnuts, and chips.6,11 It could also be used as a preservative in green foodstuffs and fishes in the same way as sodium benzoate does. CFR7 allowed the use of fumaric acid and its salts as dietetic products and nutritional additives. It can also be used as a source of available iron in the human organism if fumaric acid is combined with iron. Many fumaric acid derivatives have been approved for use in foodstuffs. 13.5.1.4 Malic Acid CFR7 allowed the use of malic acid in foodstuffs as an acidifier, aroma enhancer, and pH regulator. Malic acid also contributes to the non-browning of fruits and acts synergistically with antioxidant substances.6 Malic acid is used in the production of iced fruits, marmalades, nonalcoholic carbonated drinks as well as drinks originating from fruits. The limits of use have been defined accordingly: 3.4% nonalcoholic drinks, 35% chewing gums, 0.8% gelatins and puddings, 6.9% hard candies, 3.5% in processed fruits and juices, 3% in soft candies, and 0.7% in all other foodstuffs.


421

13.5.1.5 Tartaric Acid CFR7 allowed the use of tartaric acid in foodstuffs as an acidifier, solidifying agent, taste-enhancing agent, as a material for maintenance of humidity, and pH regulator. Tartaric acid can be used in drinks manufacturing as an enhancing agent of the red color of wine.6 It can also be used in tarts, marmalades, and candies. Mixing of tartaric acid and citric acid can be used in the production of hard candies with special flavors such as apple and wild cherry. This acid reacts with other antioxidant substances to avoid rancidity and discoloration in cheeses.6 Tartaric acid and its salts with potassium constitute ingredients of baking powders, L(+) isomer and its salts can be consumed up to 30 mg/kg body weight.9 13.5.1.6 Citric Acid Citric acid is a common metabolite of plants and animals. It is widely used in foodstuffs and pharmaceutical industry as well as the chemical industry for isolation of ions and neutralization of bases. Citric acid esters can be used as plasticizers in the manufacture of polymers and as adhesives. Citric acid can be used as an acidifier, taste enhancer, and acts synergistically with other antioxidant substances. Citric acid and its salts are used in ice creams, drinks, fruits, jams, and as an acidifier in the manufacturing of canned vegetables. Calcium citrate is used as a stabilizer in pepper, potatoes, tomato, and beans during their processing. It has also been widely applied in dairy products manufacturing.6 Moreover, it can be used in creamy sauces and soft cheeses. Sodium citrate can be used as an emulsifier. This proves to be the main acidifying factor in carbonated drinks entailing a rancid taste.6,10 It can be used in line with other substances as an antioxidant and as a decelerator of fruits’ browning. It is more hygroscopic than fumaric acid and could cause problems during storage of powdered products. Citric acid can be found in animal tissues and constitutes an intermediate product of the Krebs cycle, hence, the daily consumption levels for the acid and its salts have not been determined yet.12 13.5.1.7 Ascorbic Acid Ascorbic acid is being used as an antimicrobial and antioxidative factor and enhances the uniformity and color stability. Ascorbic acid and its salts with Na and Ca are used as food additives.7 The D isomer of ascorbic acid compared to the L isomer has no biological value and gets oxidized faster than ascorbic acid with the result of the protection of vitamin C from oxidation.10 It is used as a pH regulator to avoid the enzymatic browning of fruits and vegetables. Plants and all mammals except man, apes, and Guinea pigs synthesize ascorbic acid, hence for this reason these three need to consume alternative sources of acid such as vegetables. High doses of ascorbic acid are recommended for cancer therapy and common cold. Ascorbic acid does not cause problems if it is consumed in high doses compared to vitamins A and D that might cause problems. 13.5.1.8 Acetic Acid Acetic acid is used as a pH regulator, solvent, and as a pharmaceutical ingredient.7 It is safe when consumed in combination with the proper processing conditions. It can also be used as an additive in mustard, ketchup, mayonnaise, and sauces. Acetic acid is well known for the various unpleasant effects on humans such as allergic symptoms, ulcer,13 anaesthesia,14 and epidermic reactions15 until death.16 Acid is made up on plant and animal tissues. There is no restriction on the daily consumption of acid from humans. 13.5.1.9 Sorbic Acid Sorbic acid and its salts are widely being used as fungicides and preservatives in pickles, mayonnaise, salads, spices, fruit and vegetables pulping, jams, frozen salads, syrups, beer, wines, sweets, cheeses, yoghurt, fishes, meat, poultry, and in various bakery products.6,17 Sorbic acid is the least harmful preservative. According to a subchronic study of 2 months, carried out in 25 female and 25 male mice that consumed 40 mg acid/kg body weight, this did not lead to severe effects on the

422


TABLE 13.5 Uses and Indicated Quantities from Sorbic Acid Use Dairy products (cheese, sour cream, yoghurt) Bakery (cakes, dough, sugar crust, garnish) Vegetables (fresh salads, boiled vegetables, pickles, olives, starters) Fruits (dry fruits, fruit juices, fruit salads, jam, syrup) Drinks (wine, carbonated drinks, fruit drinks) Miscellaneous (smoked and salted fish, mayonnaise, margarine, sweets)

0.05–0.30 0.03–0.30 0.02–0.20 0.02–0.25 0.02–0.10 0.05–0.20

guinea pigs. According to studies carried out in rats fed food containing 1% or 2% sorbic acid for 80 days, no histologic abnormalities or growth problems were observed (Table 13.5).18 13.5.1.10 Propionic Acid Propionic acid is extensively used in cheeses, sweets, gelatins, puddings, jams, drinks, soft candies, Swedish cheese; at a concentration of 1%. Calcium propionate can be used as an antimicrobial agent and acts as an inhibitor of the mould formation in bread dough. No daily consumption limits were determined due to the fact that propionic acid is an intermediate metabolite of human metabolism.9 13.5.1.11 Benzoic Acid CFR7 approved benzoic acid for use at a concentration of 0.1%. The acid and its sodium salt can be used in processed foods, as food and drink preservatives with pH less than 4.5. It has an inhibitory action on mould growth and bacteria belonging to the following species: Bacillaceae, Enterobacteriaceae, and Micrococcaceae. Benzoic acid and its sodium salt can be used in the preservation of carbonated drinks and noncarbonated drinks, fruit juices, jams, mayonnaise, mustard, pickles, bakery products, and ketchup.19,20

13.5.2

COLORING AGENTS

The synthetic dyes used nowadays are divided into the following three categories: 1. FD&C dyes: Certified for food use, medicines, and cosmetics. 2. D&C dyes: Dyes considered as safe for medicinal and cosmetics use when coming into contact with muciferous tissue or when they get absorbed. 3. External D&C dyes: Dyes, which due to their toxicity do not get certified for product use due to be consumed, however, they are considered as safe for use in products of exterior applications. Moreover, coloring agents used in foodstuffs can be divided into the following two classes: 1. Dyes requiring certification 2. Dyes getting exempted from certification 13.5.2.1 Dyes Requiring Certification for Their Use 13.5.2.1.1 FD&C Red No. 2 (Amaranth) This dye was one of the first seven allowed dyes for use in 1906. Amaranth is a red brown powder easily dissolved in water and producing a deep purple or a sea red solution.


423

In experiments where amaranth was used at a percentage over 5% of the total dye, no pathological findings were reported with the exception of mutations, and tumor development in rats. Long-term experiments for 7 years in dogs injected with the above-mentioned dyes at 2% did not cause any pathological problems. Similar experiments in mice, rats, rabbits, hamsters, cats, and dogs showed neither reproduction problems nor teratogenic problems or other unpleasant effects.21 Hence, it was decided that it can be included in the list of the approved dyes. However, two experiments carried out in the ex-Soviet Union reported that the use of this dye caused carcinogenesis as well as embryotoxic reactions in mice that consumed 0.8%–1.6% of amaranth. 13.5.2.1.2 FD&C Red No. 4 This dye is allowed in foods since 1929. It is a red colored powder easily dissolved in water producing an orange-yellow solution. It was originally used as an additive in butter and margarine. It does not cause carcinogenesis in mice when contained in food at 5% for a period of 2 years. It was concluded that this dye was toxic when consumed at 1% of the food for a period of 7 years. In another experiment carried out in dogs that consumed dye at 2% for a period of 6 months, three out of four died. Due to the above-mentioned experiments the use of this dye was forbidden in 1976.22 13.5.2.1.3 Citrus Red No. 2 The use of this dye is restricted, and used mainly in leather dying. Nutritional studies showed that its use causes carcinogenesis in cats and dogs. Further experiments showed that this dye causes carcinogenesis. 13.5.2.1.4 FD&C Red No. 40 This dye is allowed in the United States and Canada following thorough experiments. However, it is not allowed in the United Kingdom, Sweden, and Europe. 13.5.2.1.5 FD&C Yellow No. 3, FD&C Yellow No. 4 These dyes are orange colored and fat soluble. They were used in 1918 in the United States as additives in margarine production. They were reported to be liver-toxic in dogs and cats.23 These dyes caused weight loss in guinea pigs even at 0.05%.24 Mice consuming a quantity of dye with their food higher than 0.05% for a period greater than 2 years showed heart atrophy or hypertrophy. Dyes are metabolized under the effect of gastric acid conditions25,26 and their metabolites caused liver cancer in rats as well as urinary bladder cancer in dogs.27 Due to these reported experimental results these dyes were rejected from the list of dyes that are allowed to be used. 13.5.2.2 Dyes Not Requiring Certification for Their Use 13.5.2.2.1 Annatto Extracts One of the older dyes used in foods, textiles, and cosmetics. The extraction of these dyes is carried out from the pericarp of the seeds of the plant Annatto tree (Bixa orellana L.). Annatto oil is produced with the distillation of the coloring agents from the seeds of Annatto tree (Bixa orellana L.) using an edible vegetable oil. The main coloring agent is norbixin (E 160b). It is supplied as an oilsoluble extract which contains mainly bixin (a mono methyl ester) or as a water-soluble product that is produced by extraction with aqueous alkali and therefore contains mainly norbixin, the free acid. Processing is primarily carried out by abrading away the pigment in a suspending agent. Abrasion may be followed by aqueous alkaline hydrolysis with simultaneous production of norbixin. Traditionally, water or vegetable oil is used as a suspending agent, although solvent processing is now also employed to produce more purified annatto extracts. Annatto is usually marketed as an extract of the annatto seed, containing amounts of the active pigments bixin or norbixin that can vary from less than 1% to over 85%.

424


In 2002, the Secretariat of the Joint FAO/WHO Expert Committee on Food Additives (JECFA) requested information relating to the toxicity, intake, and specifications of annatto extracts. Previous intake estimates for annatto had provided ambiguous results because the bixin/norbixin content of the annatto extract was unclear. As a consequence, many of the stated use levels, such as some of those appearing in the draft CCFAC General Standard for Food Additives (GSFA)28 overestimated true use levels by more than an order of magnitude. This resulted in estimates of intake of annatto extracts that appeared to exceed the JECFA ADI of 0.065 mg/kg body weight/day (based on pure bixin). Previous intake estimates for annatto provided ambiguous results because the bixin/norbixin content of annatto extract was unclear. European annatto producers consulted with the food industry to determine use levels of specific annatto extracts. These data were combined with the levels of bixin/norbixin in particular extracts to estimate the concentration of bixin/ norbixin in foods. Concentrations in food were combined with data about food consumption using various methods to estimate consumer intakes, which ranged from less than 1%–163% of the acceptable daily intake (0.065 mg/kg body weight/day). Higher intake estimates are conservative because they assume that a consumer always chooses a food that is colored with annatto extracts. In practice this is extremely unlikely, since annatto is associated only with certain product/flavor combinations.29 Food colors differ from many other food additives in that the level required to meet a technological need can vary considerably from food to food. This is because colors are usually associated with particular flavors and so the need for a coloring agent, for example an orange color such as annatto, will vary greatly depending on whether the flavor is vanilla, lemon, orange, or mango. Another source of variability is the opacity of foods. The more opaque a food is the higher the amount of color required to achieve a given shade. As a consequence, typical use levels (i.e. use in the majority of colored food products) do not necessarily reflect the maximum use level required to achieve the color density required for certain color/flavor/food combinations. It is therefore necessary to take this potential source of variability carefully into account when estimating consumer intakes. 13.5.2.2.2 Betalains (Dehydrated Sugar Beets) These dyes belong to the plants of the family Centrospermae, and more specifically plants such as beetroots and bougainvillea. Beetroot contains both dyes, the red (betacyanins) and the yellow (betaxanthins). Betalains are sensitive to light, pH, and heat.30–32 They are dissolved easily in water giving solutions of blackberry or cherry coloration. Beetroot dyes are used in foods with low storage time and do not require high and extended heat during their processing. In heat processing cases dye addition could be carried out just before or after the end of processing.30 Betalains are used at 0.1%–1% of the total dye in hard candies, yoghurt, sauces, cakes, meat substitutes, mild drinks, and gelatinous desserts. 13.5.2.2.3 Anthocyanins Anthocyanins are used as pH indicators. The dye is red at an acidic environment whereas it becomes blue as pH increases. Anthocyanins have more intense color at pH = 3.5,31 hence, for this reason these dyes are used in foods with low pH. They get discolored in the presence of amino acids33 while they get oxidized in the presence of ascorbic acid. 13.5.2.2.4 Saffron Saffron is an expensive dye since it is required to get 165,000 flower stains for the production of 50 g pure dye. The most important Saffron dyes are crocin and crocetin. Crocin has a yellow-orange color, dissolves in hot water whereas it dissolves less in alcohol. Crocetin dissolves little in water and more in organic solvents. Saffron is used for its aroma and its color is resistant to sunlight, moulds, and pH and has a high dying capacity. It is used at a concentration of 1–260 ppm in cooked food, soups, and confectionery products.


425

13.5.2.2.5 Cochineal Extracts They are derived from the body of female insects of species Coccus cacti. This insect can be found in the Canary Islands and the dye costs a great deal. It can be used in the production of candies, sweets, alcoholic and nonalcoholic drinks, jams, eye shadows, rouge, and as a coating for pills at a percentage of 0.04%–0.2%. 13.5.2.2.6 Chlorophyll Chlorophyll is the most abundant natural dye of the plants. However, it is not widely used in foods.33 Its green color is decomposed easily even at mild processing conditions. Due to its structure, during processing, magnesium gets replaced by hydrogen destroying the purple ring giving a dark brown color.34 Chlorophyll stabilizes with the replacement of magnesium ions with copper ions. Chlorophyll extracts are forbidden for use in foods in the United States. The use of complex chlorophyll-copper is allowed in the EU and Canada. 13.5.2.2.7 Carotenoids Carotenoids are sensitive to alkali and very sensitive to air, light, and high temperature. They are undissolved in water, ethyl alcohol, and glycerol. β-Carotene is one of the fewest dyes with nutritive value because it gets transformed into provitamin A in the human organism and can be used without quantitative limitations. It takes a yellow-orange color in foods when used at concentrations of 2–50 ppm. β-Carotene constitutes one of the basic ingredients of butter, margarine, cheeses, ice creams, yoghurt, and pasta products. β-Carotene (E 160 a (i)) is obtained by solvent extraction of edible plants (e.g. lucerne) or from algae (Dunaliella salina), and can be naturally accompanied by minor carotenoids (e.g. α-carotene). Nearly pure β-carotene (E 160 a (ii)) is available from the fungus Blaskeslea trispora. The coloring principle of red pepper extracts (E 160 c) is capsanthin, accompanied with capsorubin and various minor carotenoids. Lycopene (E 160 d) is found in several fruits (e.g., papaya) and represents the main carotenoid of red tomatoes. Lutein (E 161 b) is found in several plants (e.g., alfalfa) or flowers (e.g., marigold; Tagetes erecta L.). In marigold, lutein is acylated with various fatty acids. A sensitive HPLC multimethod was developed by Breithaupt35 for the determination of the carotenoid food additives (CFA) norbixin, bixin, capsanthin, lutein, canthaxanthin, b-apo-80-carotenal, b-apo-80-carotenoic acid ethyl ester, b-carotene, and lycopene in processed food using an RP C30 column. For unequivocal identification, the mass spectra of all analytes were recorded using LC–(APcI) MS. For extraction, a manual process as well as accelerated solvent extraction (ASE) was applied. Important ASE parameters were optimized. ASE was used for the first time to extract CFA from various food matrices. Average recoveries for all analytes ranged from 88.7% to 103.3% (manual extraction) and from 91.0% to 99.6% (ASE), with the exception of norbixin using ASE (67.4%). Limits of quantitation (LOQ) ranged from 0.53 to 0.79 mg/L. The presented ASE method can be used to monitor both, forbidden application of CFA or the compliance of food with legal limits. 13.5.2.2.8 Titanium Dioxide (No. 77891) This dye exists in nature in three crystalline forms—anatase, brookite, and rutile—however, only one is used as an additive). It possesses an intense white color, is resistant to sunlight, oxidation, pH, and the presence of microbes. Only the synthetically produced titanium dioxide can be used as a food additive.30 This dye cannot be dissolved in all solvents. The allowed quantity of dye in foods is up to 1% and can be used in confectionery products in the formation of white parts as well as a background. Finally, it can be used in the production of pills and cosmetics. 13.5.2.2.9 Iron Oxides and Hydroxides These synthetic dyes impart a large range of colors varying from red, yellow, and black with exceptional stability in light and temperature. Natural dyes are not acceptable as food additives due to the

426


difficulties encountered to isolate the pure substance. They are insoluble in most solvents; however, they can be dissolved in hydrogen chloride. The maximum allowed quantity of these dyes is lower than 0.25% w/w in fish paste and in pet food.

13.5.3

AROMATIC SUBSTANCES

Aroma is a food and drink property causing stimulation of the olfactory centers (Table 13.6). The role of the aromatic substances in foods is summarized below: 1. Impart a characteristic aroma, e.g. vanillin gives a vanillin aroma to ice creams 2. Enhance, complement, or modify the already existing aroma, e.g. vanillin addition to moderate chocolate or cocoa’s smell 3. Cover a non-desirable odor

TABLE 13.6 Aroma and Taste Enhancers in Food; Name, Origin, Functionality, Adverse Effects, ADI, and Applications E

Name

E256

Malic acid

E620

l-Glutamate

E621

Acidic monosodium glutamate Acidic potassium glutamate

E622

Functionality

Natural (potatoes, apples, etc.) or artificial (commercial use) Natural (amino acid of plants and animals) and artificial (commercial use) Artificial

Aromatic agent/ acidifying agent

Avoid the use of artificially made malic acid in baby foods

Aroma and taste enhancer

Its accumulation could cause damage to brain cells of guinea pigs

0–120

Taste enhancer

Its accumulation could cause brain tissue disorders High doses could cause phenomena related to high potassium It should be avoided by patients with high uric acid due to the fact of being raw material of the synthesis of uric acid Similar to E 627

0–120

Instant soups and sauces

—

Vegetable soups

—

Canned meat products, crackers, vegetable soups and sauces

Artificial

Taste enhancer

E627

Disodium Guanylate

Artificial

Taste enhancer

E631

Sodium inosinate

Natural or artificial

Taste enhancer

E635

Ribonucleic sodium

Artificial

Taste enhancer

Effects

ADIa

Origin

Similar to E 627

Product Uses Canned tomatoes, peas, orange puree with low calories, concentrated tomato juice, fruit juices Sauces, soups, aromatic lasagna and processes cheeses

Processed meta products, sauces and soups Similar to E627


427

TABLE 13.6 (continued) Aroma and Taste Enhancers in Food; Name, Origin, Functionality, Adverse Effects, ADI, and Applications E

Name

ADIa

Origin

Functionality

Effects Some people may develop nausea, vomiting, diarrhea High doses cause diarrhea and irritation

Sweets, ice creams, gums

Phenylketonuria patients should avoid it

Soft drinks, milk drinks, fruit juices, dry nuts, nutrition supplements, mustard. Low calorie products

E421

Mannitol

Natural or artificial

Sweetener and noncaking

E905

Microcrystalline wax

Artificial

E951

Aspartame

Artificial

Antifoaming agent and glazing agent Sweetener

E952

Cyclamic acid and its sodium and calcium salts

Artificial

Sweetener

Cyclamics get excreted unalterable by the human body except some others that could disrupt it and cause undesirable side effects

Product Uses

Dried fruits, citrus fruits, gums

Source: Adapted from Van den Ouweland, G.A.M. and Swaine, J.R.L., Perfumer Flavorist, 5, 15, 1980. ADI, acceptable daily intake mg/kg body weight.

a

The odor of a chemical substance has a direct relationship with its chemical formula. Compounds such as nitrogen, oxygen, and sulfur play a significant role. More specifically, the addition of a hydroxyl group into the molecule of a compound reduces or restricts its odor, whereas replacement of a hydroxyl group with a ketone group enhances the odor (Table 13.7). 13.5.3.1 Polycyclic Aromatic Hydrocarbons Polycyclic aromatic hydrocarbons (PAHs) comprise the largest class of chemical compounds known to be cancer causing agents. Some, while not carcinogenic, may also act synergically with others. PAHs are being found in water, air, soil, and, therefore also in food. They originate from diverse sources such as tobacco smoke, engine exhausts, petroleum distillates, and coal-derived products, with combustion sources predominating. However, PAHs may also form directly in food as a result of several heat processes such as grilling,36 roasting, smoke drying, and smoking. PAHs are compounds consisting of two or more condensed aromatic rings, lineated together, either cata-annellated (linearly-, or angularly), or peri-condensed. Formation, factors affecting concentrations, legal limits and occurrence of polycyclic aromatic hydrocarbons in smoked meat products and smoke flavor additives are briefly reviewed by Simko.37 The most widely employed techniques such as thin-layer chromatography (TLC), gas chromatography (GC), and high-performance liquid chromatography (HPLC) are evaluated. Moreover, sample preparation, pre-separation procedures, separation and detection systems being used for the determination have been evaluated with emphasis on the latest developments in applied food analysis and

428


TABLE 13.7 Saturated Organic Acids and Characteristics with Regard to Aroma Acid

Characteristics

Acetic Strong aroma, fine, carmine Propionic Rancid, fatty, milky Butyric Fatty, milky, rancid Valeric Rancid, sweet, chocolate Caprylic Fatty, fruit smell, soapy Myristic Soapy Palmitic Soapy, waxy Stearic Soapy, waxy Sources: WHO, Environmental Health Criteria 3: Lead, World Health Organization, Geneva, 1976; Reilly, C., Metal Contamination of Food, 2nd ed., Elsevier Applied Science, London, 1991.

TABLE 13.8 Aldehydes Found in Bovine, Chicken, and Pork Fat Aldehydes C5 C6 C7 C7 2t C7 2t 4C C8 C8 2t C9 C9 2t C9 2t 4C C10

Bovine

Chicken

Pork

+ + + + − + + + + − −

− + + + + − + + + + +

− + − + + + + + + + +

Sources: Dabeka, R.W. et al., Food Add. Contam., 89, 1987. WHO, International Standards for Drinking Waters, 2nd ed., World Health Organization, Geneva, 1963. Reilly, C., Metal Contamination of Food, 2nd ed., Elsevier Applied Science, London, 1991.

the chosen data regarding the concentration of polycyclic aromatic hydrocarbons in smoked meat products and smoke flavor additives are summarized (Table 13.8). 13.5.3.2 Flavoring Substances Munro and Danielewska-Nikiel38 conducted a study to determine the margins of safety between nonobserved-effect levels (NOELs) and estimates of daily intake for 809 flavoring substances evaluated


429

by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) between 2000 and 2004. Estimates of daily intake were calculated by means of two methods, the maximized survey-derived daily intake (MSDI) and the possible average daily intake (PADI). The MSDI estimates were based on the production volume of flavoring agents as reported by industry, whereas the higher and more conservative PADI estimates were derived by multiplying the anticipated average use level of a flavoring substance in each of the 33 food categories by the average amount of food consumed daily from that food category and summing the overall intake of the 33 food categories. These intake estimates were used to calculate the margins of safety for the flavoring agents to determine whether adequate margins of safety would still exist in the event that the MSDIs used by JECFA to evaluate the safety of flavoring substances underestimated daily intakes. Based on the calculation of the margins of safety using the MSDI values, 99.9% of the 809 flavoring substances evaluated by JECFA have margins of safety of greater than 100. In comparison, 98% of the flavoring substances have margins of safety of greater than 100 when the margins of safety were calculated according to the PADI values. The results indicate that if the MSDI estimates used by JECFA for the evaluation of the safety of flavoring substances were underestimated, a wide margin of safety exists for all but a few of the flavoring substances even when intakes were estimated from the PADI values. Although the Procedure for the Safety Evaluation of Flavoring Agents does not require toxicological data in every instance, but rather relies primarily on structure–activity relationships to assess safety, this study confirmed that on the basis of a more traditional toxicological process of evaluating chemical safety, the procedure showed that the evaluated substances did not present a concern for safety at current estimates of intake.38

13.5.4

TOXIC SUBSTANCES

It is difficult to distinguish clearly between the essential metals (micronutrients) and the toxic substances in human diet. Nearly all metals are toxic for humans if they get absorbed by the organism at high concentrations. However, it is possible to differentiate between those elements that are known definitely as essential and those that cause toxicosis at extremely low concentrations and those that do not possess known therapeutic normal functions. The most dangerous heavy metals, which occur nearly everywhere and particularly in agricultural products are mercury, lead, and cadmium. Other toxic metals are arsenic, boron, beryllium, selenium, and others. The most probable sources of food contamination with toxic substances are soil, agricultural chemicals and fertilizers, contaminated water from industrial wastes, the various food-processing stages, plants that can absorb toxic substances from the contaminated land and store them in their tissues, as well as the muddy depositions of pit wastes. The commonest metals contaminating foods are detailed below: 13.5.4.1 Lead The presence of lead in the human food chain constitutes a major hygienic problem in the world. It exists in every organ or tissue of the human body and varies between 100 and 400 mg or approximately 1.7 μg/g tissue.39 Over 90% lead is present in the bones. It constitutes a physiological ingredient of human diet (Table 13.9). The daily food uptake of lead is estimated to be approximately equal to 100–300 μg, with particularly high percentages during intense environmental pollution.40 Lead absorption contained in foods is estimated to be approximately equal to 10% for adults and 40% for children.41 Many dietetic factors seem to affect the uptake levels of lead. Hence, low calcium levels, iron deficiency, and diets rich in carbohydrates but low in proteins as well as diets containing high levels of vitamin D lead to an increased absorption of lead. In the healthy adult approximately 90% of the absorbed lead gets excreted by urine and feces. Lead infection can be distinguished as acute and chronic. Symptoms of the former include blood, nervous, gastroenteric, and hepatic failures.41 In general, phenomena such as anorexia, indigestion, and constipation occur followed by colic with intense paroxysmal abdominal pain. Although lead encephalopathy has been observed in little children42,43 very little is known regarding the symptoms

430


TABLE 13.9 Lead Concentration in Food and Drinks Food Cereals Meat and fish Fresh fruits Canned fruits Fresh vegetables Canned vegetables Milk Water Alcoholic drinks

Range (mg/kg)

Average (mg/kg)

Advances in Food Biochemistry

Food biochemistry and food processing

Food Biochemistry and Food Processing

Food Biochemistry And Food Processing

Advances in Food Research

Biochemistry of Atherosclerosis (Advances in Biochemistry in Health and Disease)

Advances in Developmental Biochemistry, Volume 3

Advances in Carbohydrate Chemistry and Biochemistry

Advances in Carbohydrate Chemistry and Biochemistry, Volume 63 (Advances in Ccarbohydrate Chemistry and Biochemistry)

Advances in Developmental Biochemistry, Volume 5

Advances in Food Protection: Focus on Food Safety and Defense

Advances in Food and Nutrition Research, Volume 52 (Advances in Food & Nutrition Research)

Advances in Food and Nutrition Research, Volume 61 (Advances in Food & Nutrition Research)

Advances in Food Research, Volume 18

Food Biotechnology (Advances in Biochemical Engineering Biotechnology)

Advances in Food Research Volume 1

Advances in Food Research, Volume 24

Advances in Food Research, Volume 28

Advances in Food Dehydration (Contemporary Food Engineering Series)

Advances in Food Research: v. 2

Advances in Food Research, Volume 17

Advances in Food Research, Volume 27

Advances in Food Research Volume 30

Advances in Food Research, Volume 21

Advances in Food Research Volume 31

Advances in Food Research, Volume 9

Advances in Food Research, Volume 24

Advances in Food Research, Volume 23

Advances in Food Research, Volume 6

Advances in Food Research. Volume 22

Advances in Food Research, Volume 6

Advances in Food Biochemistry

Food biochemistry and food processing

Food Biochemistry and Food Processing

Food Biochemistry And Food Processing

Advances in Food Research

Biochemistry of Atherosclerosis (Advances in Biochemistry in Health and Disease)

Advances in Developmental Biochemistry, Volume 3

Advances in Carbohydrate Chemistry and Biochemistry

Advances in Carbohydrate Chemistry and Biochemistry, Volume 63 (Advances in Ccarbohydrate Chemistry and Biochemistry)

Advances in Developmental Biochemistry, Volume 5

Advances in Food Protection: Focus on Food Safety and Defense

Advances in Food and Nutrition Research, Volume 52 (Advances in Food & Nutrition Research)

Advances in Food and Nutrition Research, Volume 61 (Advances in Food & Nutrition Research)

Advances in Food Research, Volume 18

Food Biotechnology (Advances in Biochemical Engineering Biotechnology)

Advances in Food Research Volume 1

Advances in Food Research, Volume 24

Advances in Food Research, Volume 28

Advances in Food Dehydration (Contemporary Food Engineering Series)

Advances in Food Research: v. 2

Advances in Food Research, Volume 17

Advances in Food Research, Volume 27

Advances in Food Research Volume 30

Advances in Food Research, Volume 21

Advances in Food Research Volume 31

Advances in Food Research, Volume 9

Advances in Food Research, Volume 24

Advances in Food Research, Volume 23

Advances in Food Research, Volume 6

Advances in Food Research. Volume 22

Advances in Food Research, Volume 6

Recommend Documents